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US9371274B2 - Compound having read-through activity - Google Patents

Compound having read-through activity Download PDF

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US9371274B2
US9371274B2 US14/291,861 US201414291861A US9371274B2 US 9371274 B2 US9371274 B2 US 9371274B2 US 201414291861 A US201414291861 A US 201414291861A US 9371274 B2 US9371274 B2 US 9371274B2
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alkynyl
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Yoshio Hayashi
Akihiro Taguchi
Fumika YAKUSHIJI
Yuri Yamazaki
Ryoichi Matsuda
Masataka Shiozuka
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University of Tokyo NUC
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Definitions

  • the present invention is a continuation-in-part application claiming priority to Japanese Patent Application 2011-263404 filed on Dec. 1, 2011 and International Application PCT/JP2012/081120 filed on Nov. 30, 2012.
  • the present invention relates to a novel compound having read-through activity.
  • the present invention also relates to a pharmaceutical composition containing the compound and use of the compound and the pharmaceutical composition to treat genetic disease.
  • Duchenne muscular dystrophy seen in male children can be given as an example of a typical genetic disease.
  • a mutation occurs on the dystrophin gene present in the X chromosome.
  • a stop codon is formed by this mutation (premature stop codon), and expression of normal dystrophin and dystrophin-associated proteins is inhibited by the interruption and cessation of translation at this mutation site.
  • dystrophin proteins are lacking, and muscular dystrophy occurs.
  • Negamycin methylhydrazinoacetic acid-linked ⁇ -hydroxy- ⁇ -lysine: NM
  • NM methylhydrazinoacetic acid-linked ⁇ -hydroxy- ⁇ -lysine
  • the purpose of the present invention is to provide a novel compound having read-through activity and a therapeutic drug for nonsense mutation-type diseases containing this compound.
  • the present inventors turned their attention to negamycin and natural analogs as regards the above problems, and, as a result of in-depth studies, succeeded in producing novel derivatives having potent read-through activity and thereby perfected the present invention.
  • Negamycin has a hydroxyl group at position 5 of the amino acids that constitute its left flank.
  • the present inventors confirmed during in-depth studies that 5-epi-negamycin, which differs from negamycin in the configuration of this position 5 hydroxyl group, also has read-through activity and discovered that this hydroxyl group does not contribute to the read-through activity.
  • Compounds not having a hydroxyl group at position 5 of the amino acids that constitute the left flank of negamycin were thus unexpectedly discovered to have the same or greater read-through activity as negamycin, and the present invention was perfected.
  • the present invention is:
  • the negamycin derivatives defined by general formula (1) of the present invention generally have better read-through activity than negamycin and are compounds useful as pharmaceutical products.
  • the present invention can thus provide pharmaceutical compositions containing the compounds defined by general formula (1).
  • the pharmaceutical compositions of the present invention can be used in the treatment or prevention of comprehensive diseases caused by nonsense mutations and in the treatment or prevention of cancers in which nonsense mutations of suppressor genes such as the p53 gene and the like participate, and are very useful.
  • FIG. 1 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
  • FIG. 2 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
  • FIG. 3 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
  • FIG. 4 is a graph showing the results of in vivo measurement of the read-through activity of compounds of the present invention.
  • FIG. 5 is a graph showing the results of in vitro measurement of the read-through activity of example compounds 3, 7, 47, and 48.
  • FIG. 6 is a graph showing the results of studying the relationship between the alkyl chain length of R 12 of an N-linked amino acid residue and the read-through activity.
  • Negamycin methylhydrazinoacetic acid-linked ⁇ -hydroxy- ⁇ -lysine
  • ⁇ -hydroxy- ⁇ -lysine is a compound represented by the following chemical formula.
  • 5-Epi-negamycin which is an epimer, also has read-through activity.
  • the present invention is based on the discovery that the configuration of this position 5 hydroxyl group does not contribute to the read-through activity.
  • the compounds of the present invention are characterized by not having a hydroxyl group at position 5 of the amino acids that constitute the left flank of negamycin.
  • the compounds of the present invention can be synthesized by: reducing a protected ⁇ -amino acid, having a protected side chain amino group, to an alcohol, and deriving a sulfonic acid ester; from here, a nitrile group is introduced and subjected to alkali hydrolysis and condensation with a hydrazine unit; finally, the protecting groups are removed and refining is conducted.
  • a compound can be obtained from commercial Boc-Orn(Boc)-OH by the reaction shown in Scheme 1 of Example 1.
  • R 1 and R 2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded, represented by the following formula (2).
  • R 8 is hydrogen, an optionally substituted C 1 -C 10 alkyl group, C 2 -C 10 alkenyl group, C 2 -C 10 alkynyl group, C 6 -C 10 aralkyl group, or C 6 -C 10 aralkenyl group;
  • Substituents that the optionally substituted C 1 -C 10 alkyl group, C 2 -C 10 alkenyl group, C 2 -C 10 alkynyl group, C 6 -C 10 aralkyl group, or C 6 -C 10 aralkenyl group in R 8 can have include a halogen, hydroxyl group, carboxyl group, amino group, amide group, or the like.
  • a preferred embodiment of the present invention is a compound represented by the following general formula (3), wherein X is N(R 3 ) (R 4 ), or a salt or solvate of the compound.
  • N-linked amino acids are ⁇ -amino acids and ⁇ -amino acids having C 1 -C 12 in a side chain, preferably one containing an amino group, cyclic amine, guanidyl group, or oxygen atom in the side chain.
  • ⁇ -amino acids More preferred as ⁇ -amino acids are leucine, isoleucine, valine, lysine, ornithine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, lysine, ⁇ -hydroxyleucine, and the like, and more preferred as ⁇ -amino acids are ⁇ -alanine and the like.
  • Chain or cyclic aliphatic hydrocarbons are also preferred as side chains consisting of C 1 -C 12 .
  • Those having a branched structure at the p or y position are more preferred, and examples include t-butylalkyl, sec-alkylalkyl, bicycloalkylalkyl groups, more specifically, t-butyl, t-butylmethyl, cyclohexylmethyl, cycloheptylmethyl, menthylmethyl, adamantylmethyl, decalinylmethyl, and the like.
  • N-linked amino acid residue of X is represented by the following formula (4).
  • R 11 is hydrogen or an optionally substituted C 1 -C 6 alkyl group
  • Examples of the substituents that the optionally substituted C 1 -C 10 alkyl group, C 2 -C 10 alkenyl group, C 2 -C 10 alkynyl group, C 6 -C 10 aralkyl group, or C 6 -C 10 aralkenyl group of R 12 can have are a halogen, hydroxyl group, carboxyl group, amino group, amide group.
  • An optionally substituted C 1 -C 10 alkyl group may be linear or branched and may form a cyclic structure.
  • Optionally substituted C 4 -C 9 alkyl groups are preferred among the optionally substituted C 1 -C 10 alkyl groups of R 12 , and optionally substituted C 7 -C 9 alkyl groups are more preferred.
  • Another preferred embodiment of the present invention is a compound represented by the following general formula (5) wherein n in formula (1) is 1, or a salt or solvate of the compound.
  • R 1 and R 2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded; this amino acid residue is represented by the above formula (2).
  • N-linked amino acid residue of X is also represented by the above formula (4).
  • Synthesis of the compounds of the present invention can be achieved easily by modifications based on the many previous reports of the total synthesis of negamycin.
  • Total synthesis examples from long ago include Y.-F. Wang, T. Izawa, S. Kobayashi and M. Ohno, J. Am. Chem. Soc., 1982, 104, 6465-6466; H. Iida, K. Kasahara and C. Kibayashi, J. Am. Chem. Soc., 1986, 108, 4647-4648, and the like. More recent examples include Davies, S. G. et al., Tetrahedron: Asymmetry 1996, 7, 1919-1922; Williams, R. M. et al., J. Org. Chem. 2002, 67, 6361-6365, and the like.
  • the present invention also includes salts of the above compounds.
  • Preferred examples of salts are pharmaceutically acceptable salts that can be used as drugs.
  • Salt hydrates and salt anhydrides are also included. Examples include salts of sodium, potassium, magnesium, calcium, aluminum, and other such inorganic bases; salts of methylamine, ethylamine, ethanolamine, and other such organic bases; salts of lysine, ornithine, and other such basic amino acids, and ammonium salts. These salts may be acid addition salts.
  • salts include acid addition salts of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and other such mineral acids; formic acid, acetic acid, propionic acid, oxalic acid malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, and other such organic acids; aspartic acid, glutamic acid, and other such acidic amino acids.
  • the present invention also includes various pharmaceutically acceptable solvates, crystalline polymorphs, and the like of hydrates and the like of the above compounds.
  • wild-type normal protein means a protein consisting of an amino acid sequence encoded by a mutation-free normal wild-type gene. Wild-type normal proteins also include proteins consisting of amino acid sequences substantially identical to the above amino acid sequence.
  • substantially identical protein means, for example, a protein having the same function and activity as the protein encoded by the normal wild-type gene that is a protein consisting of an amino acid sequence in which one or multiple amino acids have been deleted, replaced, or added in the amino acid sequence encoded by the normal wild-type gene.
  • one or multiple preferably means one or several, specifically, 1-100, preferably 1-50, more preferably 1-10, and especially 1-5, 4, 3, or 2.
  • this substantially identical protein means a protein having sequence identity of 80% or higher, preferably 90% or higher, more preferably 95% or higher, and especially 96, 97, 98, or 99% or higher with the amino acid sequence encoded by the normal wild-type gene when calculated using BLAST or the like (for example, default, i.e. initial settings, parameters) and having the same function and activity as the protein encoded by the normal wild-type gene.
  • Another separate embodiment of the present invention relates to a pharmaceutical composition containing a compound represented by the following general formula (1) or a salt or solvate of the compound.
  • 3-Epi-5-dehydroxynegamycin which was isolated from nature and known in the past is also included among compounds represented by general formula (1).
  • this natural substance could not be anticipated easily since it has weak antimicrobial activity and none of the research conducted in the past found read-through activity.
  • the presence of a natural substance having leucine added by an amide bond to the position 3 amino group was also known, but this natural substance has no antimicrobial activity and was left as a substance of unknown activity.
  • the present inventors discovered that this 3-N-leucyl-3-epi-5-dehydronegamycin also has potent read-through activity and perfected the pharmaceutical composition of the present invention.
  • One aspect of the present invention is a pharmaceutical composition containing a compound represented by the following general formula (3) or a salt or solvate of the compound.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and n are as defined in general formula (1).
  • One aspect of the present invention is a pharmaceutical composition containing a compound represented by the following general formula (5) or a salt or solvate of the compound.
  • R 1 , R 2 , X, R 3 , R 4 , R 5 , R 6 , and R 7 are as defined in general formula (1).
  • One aspect of the present invention is a pharmaceutical composition containing a compound wherein R 1 and R 2 in general formula (1), (3), or (5) are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded and this amino acid residue is represented by formula (2) below, or a salt or solvate of the compound.
  • One aspect of the present invention is a pharmaceutical composition containing a compound wherein the N-linked amino acid residue of X in general formula (1) or (5) is represented by the following formula (4), or a salt or solvate of the compound.
  • substituents that the optionally substituted C 1 -C 10 alkyl group, C 2 -C 10 alkenyl group, C 2 -C 10 alkynyl group, C 6 -C 10 aralkyl group, or C 6 -C 10 aralkenyl group of R 12 in formula (4) can have are a halogen, hydroxyl group, carboxyl group, amino group, and amide group.
  • Optionally substituted C 1 -C 10 alkyl groups may be linear or branched and may form a cyclic structure.
  • a preferred embodiment of the present invention is a pharmaceutical composition containing a compound, wherein R 12 is an optionally substituted C 4 -C 9 alkyl group, or a salt or solvate of the compound.
  • a preferred embodiment of the present invention is a pharmaceutical composition containing a compound, wherein R 12 is an optionally substituted C 7 -C 9 alkyl group, or a salt or solvate of the compound.
  • Nonsense mutation-type disease means a disease caused by the production of a stop codon midway in a gene (premature stop codon) due to a point mutation, deletion, insertion, or the like on a gene resulting in suppression of the expression of a normally functioning protein.
  • nonsense mutation-type disease means a disease caused by inhibition of the expression of a protein in association with degradation of mRNA containing a premature stop codon.
  • nonsense mutation-type diseases include muscular dystrophy, Duchenne muscular dystrophy, infantile neuronal ceroid lipofuscinosis, multiple sclerosis, Alzheimer's disease, Tay-Sachs disease, neurodegenerative disease, Parkinson's disease, and other such central nervous diseases; rheumatoid arthritis, graft-versus-host disease, and other such autoimmune diseases; arthritis and other such inflammatory diseases; hemophilia, von Willebrand disease, ataxia telangiectasia, ⁇ -thalassemia, kidney stones, and other such blood diseases; osteogenesis imperfecta, liver cirrhosis, and other such collagen diseases; Other examples include neurofibromatosis, bullous disease, lysosomal storage disease, Hurler disease, familial hypercholesterolemia, cerebellar ataxia, nodular sclerosis, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial cholesterolemia, pigmentary retinopathy, amyloidosis, atherosclerosis, gig
  • Cancer is also another example.
  • lung cancer, colorectal cancer, stomach cancer, esophageal cancer, kidney cancer, pancreatic cancer, liver cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, skin cancer, brain tumors, and the like are included as cancers in which a nonsense mutation of a p53 gene or another such suppressor gene participates.
  • it can preferably be used in muscular dystrophy, cystic fibrosis, Hurler disease, and infantile neuronal ceroid lipofuscinosis.
  • the pharmaceutical composition may contain two or more of the above compounds.
  • the compounds of the present invention can be used as a prodrug. Specifically, the water solubility of the compound decreases and the liposolubility increases when the part represented by —COOR 7 is an ester in compounds of the above formulas (1), (3), and (5).
  • Compounds of increased liposolubility promote systemic absorption and become an active form while being absorbed due to esterase present in the digestive tract and the like, increasing enteral absorption, or reach a site at which they are to act within the body and there they are converted into a carboxyl group by esterase present in the organs and tissues and can act at the site reached. As a result, the bioavailability is increased in the end.
  • the compounds of the present invention also include compounds in the form of such prodrugs and also include compounds not in the form of prodrugs.
  • the route of administration of the pharmaceutical composition of the present invention is not limited, and it can be administered by oral administration, subcutaneous administration, intracutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, per nasal administration, intra-oral administration, transmucosal administration, and the like.
  • the dosage form is also not limited. For example, tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, tinctures, syrups, extracts, and elixirs can be made for oral administration.
  • parenteral agents for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and other such injections; percutaneous administration or adhesive skin patches, ointments, or lotions; sublingual agents or oral adhesive patches for intra-oral administration; and aerosols for per nasal administration can be made.
  • a pharmaceutical composition of the present invention can be administered as an injection (for example, an intramuscular injection for direct administration to a muscle) to treat muscular dystrophy.
  • the pharmaceutical composition of the present invention may be in the form of a sustained-release or slow-release agent.
  • the pharmaceutical composition of the present invention can contain various pharmaceutically acceptable components. Examples include excipients, disintegrating agents, diluents, lubricants, flavorings, colorings, sweeteners, flavoring agents, suspending agents, wetting agents, emulsifiers, dispersing agents, auxiliary agents, preservatives, buffers, binders, stabilizers, coating agents, and the like. A plurality of these may be contained.
  • the dose of the pharmaceutical composition of the present invention is not limited, but is selected as is appropriate in accordance with the efficacy of the components contained, the dosage form, route of administration, type of disease, weight, age, medical conditions, and other such characteristics of the patient being administered, the judgment of a physician, and the like.
  • it is a range of approximately 0.01 ⁇ g to approximately 100 mg, preferably approximately 0.1 ⁇ g to approximately 1 mg, per kilogram of the patient's body weight.
  • the dose can be administered from once to divided over several times per day or may be administered intermittently once every several days or every several weeks.
  • the read-through activity of compounds of the present invention can be tested, for example, by constructing a vector containing (i) a promoter, (ii) a first translation initiation codon and first reporter gene positioned downstream of the above promoter, (iii) a second translation initiation codon and second reporter gene positioned downstream of the above first reporter gene, (iv) a sequence containing a premature stop codon from a causative gene of a nonsense mutation-type disease positioned between the above first reporter gene and second translation initiation codon, and (v) a translation stop codon positioned downstream of the above second reporter gene, introducing this vector into host cells or creating transgenic animals with the vector introduced into animals, and using these host cells or transgenic animals.
  • a compound is administered to the host cells or transgenic animals, and the ratio of the expression level of the second reporter gene to the first reporter gene is compared between a case with and a case without addition of the test compound.
  • a CMV promoter, ⁇ -actin promoter, or the like can be used as the promoter.
  • a ⁇ -galactosidase gene, luciferase gene, GFP (green fluorescent protein) gene, CAT (chloramphenicol acetyl transferase) gene, or the like can be used as the first reporter gene and second reporter gene.
  • a sequence containing a stop codon (TAA, TAG, or TGA) in a reading frame may be used as the sequence containing a premature stop codon from the causative gene of a nonsense-type disease.
  • a gene containing a premature stop codon of a dystrophin gene of mdx mice may be used.
  • Example compound 2 was synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • N-methylmorpholine (336 ⁇ L, 3.01 mmol) and isobutyl chloroformate (395 ⁇ L, 3.01 mmol) were added to a THF (3 mL) solution of Boc-Orn(Boc)-OH (1.00 g, 3.01 mol) at ⁇ 15° C. and stirred for 10 minutes at the same temperature.
  • the reaction solution was filtered, washed with THF, and a water (1.5 mL) solution of sodium borohydride (171 mg, 4.52 mmol) was added to the mother liquor obtained in an ice-salt bath and stirred for 20 minutes at the same temperature.
  • Example compound 5 was synthesized by the following synthesis scheme.
  • N-methylmorpholine (1.97 mL, 15.0 mmol) and isobutyl chloroformate (1.68 mL, 15.0 mmol) were added to a THF (14 mL) solution of Boc-Orn(Cbz)-OH (5.00 g, 13.6 mmol) at ⁇ 15° C. and stirred for 10 minutes at the same temperature.
  • the reaction solution was filtered and washed with THF.
  • a water (4 mL) solution of sodium borohydride (772 mg, 20.4 mmol) was added to the mother liquor obtained in an ice-salt bath and stirred for 10 minutes at the same temperature. Water was added to the reaction solution while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na 2 SO 4 . After filtration, a colorless oily substance was obtained by distilling off the mother liquor under reduced pressure. This was used in the next reaction without refining.
  • Diisobutyl aluminum hydride (1.00 M toluene solution, 47.7 mL, 47.7 mmol) was added dropwise at ⁇ 78° C. to an anhydrous dichloromethane (80 mL) solution of compound 6 (5.74 g, 15.9 mmol) under an argon atmosphere. The temperature of the reaction solution was raised to ⁇ 50° C., and it was stirred for 1 hour 20 minutes. Methanol and saturated sodium potassium tartrate aqueous solution were added to the reaction solution at the same temperature, and the temperature was raised to room temperature, followed by extraction by chloroform. The organic layer was washed with saturated saline and dried by Na 2 SO 4 .
  • a colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H 2 O in 0.1% TFA: CH 3 CN in 0.1% TFA. A linear gradient of 5-10% CH 3 CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (19.3 mg, 39.6 ⁇ mol, two steps, 44%)
  • a colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H 2 O in 0.1% TFA: CH 3 CN in 0.1% TFA. A linear gradient of 10-20% CH 3 CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (26.7 mg, 56.2 ⁇ mol, two steps, 54%)
  • Example compound 7 was synthesized by the following synthesis scheme.
  • reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate.
  • the organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na 2 SO 4 .
  • a white, cottony substance was obtained by refining by high-performance liquid chromatography (solvent, H 2 O in 0.1% TFA: CH 3 CN in 0.1% TFA. A linear gradient of 10-15% CH 3 CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (16.1 mg, 28.0 ⁇ mol, two steps, 60%)
  • Example compound 3 was synthesized by the following synthesis scheme.
  • 18-Crown-6 (2.76 g, 10.4 mmol) and potassium cyanide (2.72 g, 41.7 mmol) were added to an acetonitrile (105 mL) solution of compound 16 (7.97 g, 20.9 mmol) and stirred overnight at 40° C. After the reaction solution had returned to room temperature, saturated sodium bicarbonate aqueous solution was added while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na 2 SO 4 .
  • reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate.
  • the organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na 2 SO 4 .
  • Example compounds 14-18 were synthesized by the following synthesis scheme. Furthermore, in the scheme, the numbers appended below the structural formulas represent the compound numbers.
  • Compound 20b was synthesized by the same method as compound 14 using compound 13 (61.2 mg, 0.110 mmol) (colorless solid, 39.3 mg, 54.6 ⁇ mol, two steps 50%).
  • Compound 20c was synthesized by the same method as compound 14 using 13 (71.7 mg, 0.129 mmol) (white solid, 77.8 mg, 0.119 mmol, two steps, 92%).
  • Compound 20d was synthesized by the same method as compound 14 using compound 13 (52.0 mg, 93.5 ⁇ mol) (colorless solid, 51.7 mg, 84.3 ⁇ mol, two steps 90%).
  • Example compounds 19-20 were synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • Compound 22b was synthesized by the same method as compound 22a using compound 17 (111 mg, 0.354 mmol) and H 2 N—N(Me)CH 2 CO 2 Bn(o-Br) (62.6 mg, 0.229 mmol) (white solid, 75.4 mg, 0.128 mmol, two steps, 36%).
  • Example compounds 21-24 were synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • Compound 24b was synthesized by the same method as compound 24a using compound 22a (120 mg, 0.236 mmol) and 4-bromobenzyl alcohol (53.0 mg, 0.283 mmol) (white solid, 71.6 mg, 0.122 mmol, two steps, 52%).
  • Compound 24c was synthesized by the same method as compound 24a using compound 22a (99.6 mg, 0.196 mmol) and 2-nitrobenzyl alcohol (36.0 mg, 0.235 mmol) (white solid, 28.3 mg, 51.1 ⁇ mol, two steps, 26%).
  • Example compounds 25-28 were synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • Acetone (4 mL) and esterase from porcine liver were added to a phosphate buffer solution (pH 8, 0.5 M, 100 mL) of compound 27 (1.00 g, 3.60 mmol), maintained at pH 8 at 25° C., and stirred overnight.
  • Concentrated hydrochloric acid was added to the reaction solution to bring the pH close to 1, and the solution was filtered. After extracting by chloroform and washing the organic layer with saturated saline and drying (Na 2 SO 4 ), the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining.
  • Triethylamine (42.1 ⁇ L, 0.303 mmol) and methanesulfonyl chloride (31.1 ⁇ L, 0.404 mmol) were added while cooling by ice and stirring to a dichloromethane solution (1 mL) of compound 28 (50.0 mg, 0.202 mmol) and stirred for 2 hours minutes at room temperature.
  • Water was added to the reaction solution while cooling by ice and stirring, followed by extraction by chloroform. After washing the organic layer with saturated saline and drying (Na 2 SO 4 ), the solvent was distilled off under reduced pressure.
  • PTSA.H 2 N—N(Me)CH 2 CO 2 t-Bu (1.22 g, 3.68 mmol) and HOBt.H 2 O (564 mg, 3.68 mmol) were added to a DMF solution (9 mL) of the residue, triethylamine (510 ⁇ L, 3.68 mmol) and EDC.HCl (705 mg, 3.68 mmol) were added while cooling by ice and stirring, and stirred overnight at room temperature.
  • the reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate.
  • Example compounds 29-31 were synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • Example compound 32 was synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • Example compounds 33-46 were synthesized by the following synthesis scheme.
  • the numbers appended below the structural formulas represent the compound numbers.
  • reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate.
  • the organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline, and dried by Na 2 SO 4 .
  • a white solid (10.4 mg, 17.5 ⁇ mol, two steps, 24%) was obtained by refining by high-performance liquid chromatography (solvent, H 2 O in 0.1% TFA: CH 3 CN in 0.1% TFA. A linear gradient of 10-15% CH 3 CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV).
  • Triethylamine (51.8 ⁇ L, 0.372 mmol) and EDC.HCl (71.3 mg, 0.372 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature.
  • the reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate.
  • a white cottony substance was obtained by refining by high-performance liquid chromatography (solvent, H 2 O in 0.1% TFA: CH 3 CN in 0.1% TFA. A linear gradient of 40-50% CH 3 CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (5.52 mg, 8.77 ⁇ mol, two steps, 29%)
  • Example compound 49 was synthesized by the same method as compound 24a, example compound 20 using compound 22a (100 mg, 0.197 mmol) and 3-chlorobenzyl alcohol (34.4 mg, 0.242 mmol) (white solid, 14.0 mg, three steps, 49%).
  • African green monkey SV40-transformed kidney fibroblast cell line (COS-7) was sown in 100 ⁇ 20 mm cell culture dishes manufactured by Falcon and cultured at 37° C. in 5% CO 2 . Fifty milliliters of fetal bovine serum (FBS) was added to 500 mL of D-MEM (containing high glucose, L-glutamine, and phenol red) manufactured by Wako Pure Chemical Industries Ltd., and culture broth prepared to adjust to 10% FBS was used as the culture broth. (Referred to hereinafter as culture broth.)
  • D-MEM containing high glucose, L-glutamine, and phenol red
  • the Reporter Lysis Buffer 5 ⁇ , assay 2 ⁇ buffer, 1 M Na 2 CO 3 , and standard used in creation of the calibration curve referred to hereinafter are included in the ⁇ -Galactosidase Enzyme Assay System with Reporter Lysis Buffer manufactured by Promega.
  • COS-7 cells (8.0 ⁇ 10 3 cells/well) were seeded by continuous dispensing of 100 ⁇ L each into flat-bottom 96-well plates (Costar (registered trade mark) 3596) manufactured by Corning and incubated for 15-16 hours at 37° C.
  • a quantity of 4 ⁇ L/well of DNA solution prepared in a ratio of 1.32 ⁇ L (2 ⁇ g) of reporter gene (a construct obtained by linking a ⁇ -galactosidase gene and a luciferase gene and inserting a premature stop codon (TGA) at their juncture), 100 ⁇ L of OPTI-MEM (registered trade mark) I (Reduced Serum Medium 1 ⁇ ) manufactured by Invitrogen, and 4 ⁇ L of FuGENE (registered trade mark) HD Transfection Reagent manufactured by Roche was added to the solution and incubated for 10-11 hours at 37° C.
  • reporter gene a construct obtained by linking a ⁇ -galactosidase gene and a luciferase gene and inserting a premature stop codon (TGA) at their juncture
  • OPTI-MEM registered trade mark
  • I Reduced Serum Medium 1 ⁇
  • FuGENE registered trade mark
  • the entire culture broth was aspirated, and the compound to be evaluated was prepared to adjust to 200 ⁇ M by culture broth and added by continuously dispensing 200 ⁇ L/well. 100 ⁇ M and 50 ⁇ M were also prepared in the same way and added by continuously dispensing 200 ⁇ L each. Incubation was carried out for 48 hours at 37° C. after addition.
  • the entire culture broth in the well was aspirated and washed twice with PBS.
  • the Reporter Lysis Buffer 5 ⁇ was then diluted by MilliQ, and a solution prepared to adjust to Reporter Lysis Buffer l ⁇ was added by continuously dispensing 100 ⁇ L each and allowed to stand for 15 minutes at room temperature. The contents of each well were recovered thereafter, and the entire amount was transferred to Nunc microwell plates (V bottom) manufactured by Thermo Fisher Scientific and centrifuged (1800 rpm, 15 minutes). Eighty-five microliters of this lysate was transferred to Nunc low-binding 96-well plates (flat bottom) clear manufactured by Thermo Fisher Scientific.
  • a quantity of 50 ⁇ L/well of the recovered lysate was added to 96-well white plates (Costar (registered trade mark) 3912) manufactured by Corning and incubated for five minutes at room temperature after adding 100 ⁇ L of PicaGene (registered trade mark) manufactured by Toyo Ink Co., Ltd. After five minutes, the emission intensity was measured by a Berthold luminometer MicroLumat Plus LB96V manufactured by Berthold Japan.
  • Example compound 1 The read-through activity was evaluated using example compound 2 synthesized in Example 1, example compound 3 synthesized in Example 5, and example compound 1 and example compound 4 synthesized separately by the same method. The results are shown in FIG. 1 . It is understood from FIG. 1 that example compounds 1-4 have read-through activity in the same way as negamycin, and that example compound 3 in particular has remarkably high read-through efficiency.
  • FIG. 2 shows the results obtained by evaluating the read-through activity of these compounds, example compound 2, example compound 3, and negamycin.
  • FIG. 2 shows that example compounds 2, 3, 5, and 7 have remarkably high read-through efficiency in comparison even to negamycin.
  • FIG. 3 shows that these compounds which have fewer carbon atoms in the main chain than example compound 2 also have read-through efficiency equal to or greater than that of negamycin.
  • Example compounds 14-46 obtained in Examples 6-12 was evaluated by the same method as in Example 16. The results are shown below in Table 1.
  • Example Compound Read-through Activity 14 2.13 ⁇ 0.08 15 1.92 ⁇ 0.21 16 1.87 ⁇ 0.29 17 2.10 ⁇ 0.90 18 1.30 ⁇ 0.09 19 3.25 ⁇ 0.07 20 7.07 ⁇ 0.45 21 7.04 ⁇ 0.14 22 5.18 ⁇ 0.04 23 5.65 ⁇ 0.09 24 3.89 ⁇ 0.16 25 1.26 ⁇ 0.07 26 1.44 ⁇ 0.04 27 1.64 ⁇ 0.12 28 1.40 ⁇ 0.04 29 2.84 ⁇ 0.06 30 1.70 ⁇ 0.06 31 1.75 ⁇ 0.02 32 1.24 ⁇ 0.09 33 2.34 ⁇ 0.11 34 2.00 ⁇ 0.13 35 3.09 ⁇ 0.62 36 1.92 ⁇ 0.40 37 2.78 ⁇ 0.67 38 1.75 ⁇ 0.19 39 1.63 ⁇ 0.14 40 1.50 ⁇ 0.05 41 1.57 ⁇ 0.05 42 2.14 ⁇ 0.18 43 2.20 ⁇ 0.05 44 1.58 ⁇ 0.05 45 1.65 ⁇ 0.18 46 1.60 ⁇ 0.13
  • Transgenic mice were produced by introducing a luciferase gene and ⁇ -galactosidase gene as reporter genes by the method described in International Publication WO2008/004610 pamphlet.
  • Example compound 2 synthesized in Example 1 was administered subcutaneously to the abdomen of the transgenic mice produced every 24 hours for seven consecutive days. On the eighth day, the rectus femoris and the gastrosoleus muscle of the mice were excised. After mincing by ophthalmological scissors, three times the wet weight of Reporter Lysis Buffer (Promega, US) was added, and the tissue was disrupted by Physcotron (Niti-on Co., Ltd.).
  • the supernatant obtained by centrifugation was introduced into a 96-well plate, and the ⁇ -galactosidase activity was measured by a Beta-Glo Assay System (Promega, US) and the luciferase activity was measured by a Bright-Glo Assay System (Promega, US).
  • a luminometer (ATTO) was used in measurement. The luciferase activity value was divided by the ⁇ -galactosidase activity value, and the value multiplied by 10 4 was taken as the read-through efficiency.
  • FIG. 4 shows the results of measurement of the read-through activity of gentamicin, 5-epi-negamycin, and example compound 2 of the present invention.
  • Example compound 2 of the present invention was found to have read-through activity and had high read-through activity even in comparison to gentamicin and 5-epi-negamycin.

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Abstract

[Problem] Provision of a novel compound having read-through activity and a drug for the treatment of nonsense mutation-type disease containing this compound.
[Solution] A compound represented by the following general formula (1):
Figure US09371274-20160621-C00001
and a pharmaceutical composition containing this compound.

Description

The present invention is a continuation-in-part application claiming priority to Japanese Patent Application 2011-263404 filed on Dec. 1, 2011 and International Application PCT/JP2012/081120 filed on Nov. 30, 2012.
TECHNICAL FIELD
The present invention relates to a novel compound having read-through activity. The present invention also relates to a pharmaceutical composition containing the compound and use of the compound and the pharmaceutical composition to treat genetic disease.
BACKGROUND ART
In genetic disease, abnormalities occur in genes for various reasons and can interfere with life activities. Abnormalities among them due to nonsense mutations, because the stop codon occurs in the middle of a structural gene, suppress expression of a full-length protein having a function, triggering various genetic diseases. There are said to be 2500 or more nonsense mutation-type genetic diseases. Replacement therapy is exceptionally successful in hormone deficiency due to genetic abnormality, but there generally is not yet any direct method of treating genetic diseases. We currently rely on symptomatic treatment to alleviate symptoms, and no treatment leading to a complete cure has been discovered. Gene therapy is one of the most promising treatments, but this treatment has not reached a stage that can withstand clinical application.
Duchenne muscular dystrophy seen in male children can be given as an example of a typical genetic disease. In this disease, a mutation occurs on the dystrophin gene present in the X chromosome. A stop codon is formed by this mutation (premature stop codon), and expression of normal dystrophin and dystrophin-associated proteins is inhibited by the interruption and cessation of translation at this mutation site. As a result, dystrophin proteins are lacking, and muscular dystrophy occurs.
The use of compounds having read-through activity to treat nonsense mutation-type diseases has been reported. When a specific compound is administered to a patient having a premature stop codon due to a nonsense mutation and lacking a specific protein, a phenomenon is seen whereby the compound acts on the ribosomes and the ribosomes read through and translate the stop codon. This phenomenon is called read-through. As a result of read-through, a wild-type normal protein is synthesized, and the disease can be treated. Gentamicin, which is an aminoglycoside antibiotic, is known as a compound having such read-through activity. When gentamicin is administered to a Duchenne muscular dystrophy patient, dystrophin proteins are reported to accumulate (see Non-patent Reference 1). It is also reported that the typical electrophysiological abnormalities can be normalized by administering locally to the airway epithelium of a cystic fibrosis patient (see Non-patent Reference 2). However, gentamicin, like other aminoglycoside antibiotics, has severe renal toxicity and ototoxicity and is unsuited to this treatment method which will likely require long-term administration. It is also necessary to separate the drug effect from the antimicrobial activity that this compound has in consideration of adverse effects and the like.
Negamycin (methylhydrazinoacetic acid-linked δ-hydroxy-β-lysine: NM), a dipeptide antibiotic, is also reported to have read-through activity. When negamycin is administered to muscular dystrophy model mice, the expression of dystrophin is reported to recover (see Patent Reference 1). However, since negamycin also has high antimicrobial activity, it is desirable in consideration of adverse effects to provide a therapeutic drug for nonsense mutation-type diseases having selective read-through activity.
PRIOR ART REFERENCES Patent References
  • Patent Reference 1: International Publication WO2002/102361 pamphlet
NON-PATENT REFERENCES
  • Non-Patent Reference 1: Acta. Myol., 22, p. 15-21 (2003
  • Non-Patent Reference 2: N. Engl. J. Med., 349, p. 1433-1441 (2003)
  • Non-Patent Reference 3: Y. F. Wang et al., J. Am. Chem. Soc., 104, p. 6465-6466 (1982)
  • Non-Patent Reference 4: H. Iida et al., J. Am. Chem. Soc., 108, p. 4647-4648 (1986)
SUMMARY OF THE INVENTION Problems to be Solved by the Invention
The purpose of the present invention is to provide a novel compound having read-through activity and a therapeutic drug for nonsense mutation-type diseases containing this compound.
Means Used to Solve the Above-Mentioned Problems
The present inventors turned their attention to negamycin and natural analogs as regards the above problems, and, as a result of in-depth studies, succeeded in producing novel derivatives having potent read-through activity and thereby perfected the present invention.
Negamycin has a hydroxyl group at position 5 of the amino acids that constitute its left flank. The present inventors, however, confirmed during in-depth studies that 5-epi-negamycin, which differs from negamycin in the configuration of this position 5 hydroxyl group, also has read-through activity and discovered that this hydroxyl group does not contribute to the read-through activity. Compounds not having a hydroxyl group at position 5 of the amino acids that constitute the left flank of negamycin were thus unexpectedly discovered to have the same or greater read-through activity as negamycin, and the present invention was perfected.
Specifically, the present invention is:
    • (1) A compound represented by the following general formula (1):
Figure US09371274-20160621-C00002
in which
    • R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
    • R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, and where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group,
    • R1 and R2 also may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
    • X is N(R3) (R4), an N-linked amino acid residue, or a C1-C6 alkyl group containing an amino group having a substituent, where the substituent of this amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, and where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R5 is a C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
    • R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of the substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, sulfonic acid group; and
    • n is an integer of 0-3,
      or a salt or solvate of the compound,
    • (2) The compound according to (1) represented by the following general formula (3):
Figure US09371274-20160621-C00003
    • in which R1, R2, R3, R4, R5, R6, R7, and n are as defined in (1),
      or a salt or solvate of the compound,
    • (3) The compound according to (1), wherein the N-linked amino acid residue of X is selected from α-amino acids and β-amino acids,
      or a salt or solvate of the compound,
    • (4) The compound according to (1) represented by the following general formula (5):
Figure US09371274-20160621-C00004
    • in which R1, R2, X, R3, R4, R5, R6, and R7 are as defined in (1),
      or a salt or solvate thereof,
    • (5) The compound according to (1) or (4), wherein R1 and R2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded, and the amino acid residue is represented by the following formula (2):
Figure US09371274-20160621-C00005
in which
    • R8 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
    • R9, R10 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group,
    • R9 and R10 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group; and
    • m is an integer of 0-4,
      or a salt or solvate thereof,
    • (6) The compound according to any of (1), (4), and (5), wherein the N-linked amino acid residue of X is represented by the following formula (4):
Figure US09371274-20160621-C00006
in which
    • R11 is hydrogen or an optionally substituted C1-C6 alkyl group;
    • R12 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, where the substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
    • R13, R14 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R8 and R9 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members (for example, oxazolidine), where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, or C6-C10 aralkenyl group; and
    • p is an integer of 0-3,
      or a salt or solvate thereof,
    • (7) A pharmaceutical composition containing a compound represented by the following general formula (1):
Figure US09371274-20160621-C00007
in which
    • R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
    • R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group,
    • R1 and R2 also may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
    • X is N(R3) (R4), an N-linked amino acid residue, or C1-C6 alkyl group containing an amino group having a substituent, where the substituent of the amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
    • R5 is a C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
    • R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
    • R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, C3-C7 cycloalkyl group, where the substituent of a substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, sulfonic acid group; and
    • n is an integer of 0-3
      or a salt or solvate of the compound,
    • (8) The pharmaceutical composition according to (7) containing a compound represented by the following general formula (5):
Figure US09371274-20160621-C00008
    • in which R1, R2, X, R3, R4, R5, R6, and R7 are as defined in (7),
      or a salt or solvate of the compound,
    • (9) The pharmaceutical composition according to (7) or (8) for use in the treatment or prevention of a disease caused by a nonsense mutation,
    • (10) The pharmaceutical composition according to (9), wherein the disease caused by a nonsense mutation is a disease selected from the group consisting of muscular dystrophy, Duchenne muscular dystrophy, infantile neuronal ceroid lipofuscinosis, multiple sclerosis, Alzheimer's disease, Tay-Sachs disease, neurodegenerative disease, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis, hemophilia, von Willebrand disease, ataxia telangiectasia, β-thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler disease, familial hypercholesterolemia, cerebellar ataxia, nodular sclerosis, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial cholesterolemia, pigmentary retinopathy, amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, Niemann-Pick disease, and Marfan syndrome, and
    • (11) The pharmaceutical composition according to (10), wherein the disease caused by a nonsense mutation is a disease selected from the group consisting of muscular dystrophy, cystic fibrosis, Hurler disease, and infantile neuronal ceroid lipofuscinosis.
Advantages of the Invention
The negamycin derivatives defined by general formula (1) of the present invention generally have better read-through activity than negamycin and are compounds useful as pharmaceutical products. The present invention can thus provide pharmaceutical compositions containing the compounds defined by general formula (1). The pharmaceutical compositions of the present invention can be used in the treatment or prevention of comprehensive diseases caused by nonsense mutations and in the treatment or prevention of cancers in which nonsense mutations of suppressor genes such as the p53 gene and the like participate, and are very useful.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
FIG. 2 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
FIG. 3 is a graph showing the results of in vitro measurement of the read-through activity of compounds of the present invention.
FIG. 4 is a graph showing the results of in vivo measurement of the read-through activity of compounds of the present invention.
FIG. 5 is a graph showing the results of in vitro measurement of the read-through activity of example compounds 3, 7, 47, and 48.
FIG. 6 is a graph showing the results of studying the relationship between the alkyl chain length of R12 of an N-linked amino acid residue and the read-through activity.
 BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is explained in detail below. Negamycin (methylhydrazinoacetic acid-linked δ-hydroxy-β-lysine) is a compound represented by the following chemical formula.
Figure US09371274-20160621-C00009
5-Epi-negamycin, which is an epimer, also has read-through activity. The present invention is based on the discovery that the configuration of this position 5 hydroxyl group does not contribute to the read-through activity. The compounds of the present invention are characterized by not having a hydroxyl group at position 5 of the amino acids that constitute the left flank of negamycin.
Their structural formula is shown below.
Figure US09371274-20160621-C00010
The compounds of the present invention can be synthesized by: reducing a protected α-amino acid, having a protected side chain amino group, to an alcohol, and deriving a sulfonic acid ester; from here, a nitrile group is introduced and subjected to alkali hydrolysis and condensation with a hydrazine unit; finally, the protecting groups are removed and refining is conducted. For example, a compound can be obtained from commercial Boc-Orn(Boc)-OH by the reaction shown in Scheme 1 of Example 1.
Compounds according to the present invention are compounds represented by the following general formula (1) or salts or solvates thereof.
Figure US09371274-20160621-C00011
in which
    • R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
    • R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group,
    • R1 and R2 also may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
    • X is N(R3) (R4), an N-linked amino acid residue, or C1-C6 alkyl group containing an amino group having a substituent, where the substituent of the amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R5 is a C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
    • R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group,
    • R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group (benzyl group, phenethyl group, or the like), or C3-C7 cycloalkyl group, where the substituent of a substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, sulfonic acid group;
    • n is an integer of 0-3.
In a preferred embodiment in the present invention, R1 and R2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded, represented by the following formula (2).
Figure US09371274-20160621-C00012
In formula (2), R8 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
    • R9 and R10 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R9 and R10 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members (for example, oxazolidine and the like), where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • m is an integer of 0-4.
Substituents that the optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group in R8 can have include a halogen, hydroxyl group, carboxyl group, amino group, amide group, or the like.
A preferred embodiment of the present invention is a compound represented by the following general formula (3), wherein X is N(R3) (R4), or a salt or solvate of the compound.
Figure US09371274-20160621-C00013
    • in which R1, R2, R3, R4, R5, R6, R7, and n are as defined in general formula (1).
Another preferred embodiment of the present invention is a compound represented by formula (1), wherein X is an N-linked amino acid residue, or a salt or solvate of the compound. Here, examples of N-linked amino acids are α-amino acids and β-amino acids having C1-C12 in a side chain, preferably one containing an amino group, cyclic amine, guanidyl group, or oxygen atom in the side chain. More preferred as α-amino acids are leucine, isoleucine, valine, lysine, ornithine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, lysine, α-hydroxyleucine, and the like, and more preferred as β-amino acids are β-alanine and the like. Chain or cyclic aliphatic hydrocarbons are also preferred as side chains consisting of C1-C12. Those having a branched structure at the p or y position are more preferred, and examples include t-butylalkyl, sec-alkylalkyl, bicycloalkylalkyl groups, more specifically, t-butyl, t-butylmethyl, cyclohexylmethyl, cycloheptylmethyl, menthylmethyl, adamantylmethyl, decalinylmethyl, and the like.
In a preferred aspect of the present invention, the N-linked amino acid residue of X is represented by the following formula (4).
Figure US09371274-20160621-C00014
In formula (4), R11 is hydrogen or an optionally substituted C1-C6 alkyl group;
    • R12 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
    • R13, R14 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R8 and R9 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members (for example, oxazolidine), where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • p is an integer of 0-3.
Examples of the substituents that the optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group of R12 can have are a halogen, hydroxyl group, carboxyl group, amino group, amide group. An optionally substituted C1-C10 alkyl group may be linear or branched and may form a cyclic structure.
Optionally substituted C4-C9 alkyl groups are preferred among the optionally substituted C1-C10 alkyl groups of R12, and optionally substituted C7-C9 alkyl groups are more preferred.
In formula (4), when p is 0, the amino acid residue linked to N takes the form of an α-amino acid; when p is 1, it takes the form of a β-amino acid. Those preferred as α-amino acids and β-amino acids introduced by linking to N in formula (4) are the same as the preferred α-amino acids and β-amino acids listed above.
Another preferred embodiment of the present invention is a compound represented by the following general formula (5) wherein n in formula (1) is 1, or a salt or solvate of the compound.
Figure US09371274-20160621-C00015
In a preferred embodiment of compounds of formula (3), R1 and R2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded; this amino acid residue is represented by the above formula (2).
In a preferred embodiment of compounds of formula (3), the N-linked amino acid residue of X is also represented by the above formula (4).
Compounds of the following formulas (6)-(18) are given as specific examples of compounds of the present invention, but are not limited thereto.
Figure US09371274-20160621-C00016
Figure US09371274-20160621-C00017
Figure US09371274-20160621-C00018
Figure US09371274-20160621-C00019
Figure US09371274-20160621-C00020
Figure US09371274-20160621-C00021
Figure US09371274-20160621-C00022
Figure US09371274-20160621-C00023
Figure US09371274-20160621-C00024
Figure US09371274-20160621-C00025
Figure US09371274-20160621-C00026
Synthesis of the compounds of the present invention can be achieved easily by modifications based on the many previous reports of the total synthesis of negamycin. Total synthesis examples from long ago include Y.-F. Wang, T. Izawa, S. Kobayashi and M. Ohno, J. Am. Chem. Soc., 1982, 104, 6465-6466; H. Iida, K. Kasahara and C. Kibayashi, J. Am. Chem. Soc., 1986, 108, 4647-4648, and the like. More recent examples include Davies, S. G. et al., Tetrahedron: Asymmetry 1996, 7, 1919-1922; Williams, R. M. et al., J. Org. Chem. 2002, 67, 6361-6365, and the like.
The present invention also includes salts of the above compounds. Preferred examples of salts are pharmaceutically acceptable salts that can be used as drugs. Salt hydrates and salt anhydrides are also included. Examples include salts of sodium, potassium, magnesium, calcium, aluminum, and other such inorganic bases; salts of methylamine, ethylamine, ethanolamine, and other such organic bases; salts of lysine, ornithine, and other such basic amino acids, and ammonium salts. These salts may be acid addition salts. Concrete examples of such salts include acid addition salts of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and other such mineral acids; formic acid, acetic acid, propionic acid, oxalic acid malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, and other such organic acids; aspartic acid, glutamic acid, and other such acidic amino acids.
The present invention also includes various pharmaceutically acceptable solvates, crystalline polymorphs, and the like of hydrates and the like of the above compounds.
The compounds of the present invention act on the ribosomes in a situation in which a premature stop codon is produced by a nonsense mutation and a specific protein cannot be produced; the ribosomes read through the premature stop codon produced by the nonsense mutation and carry out translation, resulting in the production of a wild-type normal protein. In the present invention, the term wild-type normal protein means a protein consisting of an amino acid sequence encoded by a mutation-free normal wild-type gene. Wild-type normal proteins also include proteins consisting of amino acid sequences substantially identical to the above amino acid sequence. The term substantially identical protein means, for example, a protein having the same function and activity as the protein encoded by the normal wild-type gene that is a protein consisting of an amino acid sequence in which one or multiple amino acids have been deleted, replaced, or added in the amino acid sequence encoded by the normal wild-type gene. Here, one or multiple preferably means one or several, specifically, 1-100, preferably 1-50, more preferably 1-10, and especially 1-5, 4, 3, or 2. Moreover, this substantially identical protein means a protein having sequence identity of 80% or higher, preferably 90% or higher, more preferably 95% or higher, and especially 96, 97, 98, or 99% or higher with the amino acid sequence encoded by the normal wild-type gene when calculated using BLAST or the like (for example, default, i.e. initial settings, parameters) and having the same function and activity as the protein encoded by the normal wild-type gene.
Another separate embodiment of the present invention relates to a pharmaceutical composition containing a compound represented by the following general formula (1) or a salt or solvate of the compound.
Figure US09371274-20160621-C00027
in which
    • R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
    • R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group,
    • R1 and R2 also may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
    • X is N(R2)(R4), an N-linked amino acid residue, or C1-C6 alkyl group containing an amino group having a substituent, where the substituent of the amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, here, R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R5 is a C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
    • R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of a substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, sulfonic acid group;
    • n is an integer of 0-3.
3-Epi-5-dehydroxynegamycin which was isolated from nature and known in the past is also included among compounds represented by general formula (1). However, this natural substance could not be anticipated easily since it has weak antimicrobial activity and none of the research conducted in the past found read-through activity. The presence of a natural substance having leucine added by an amide bond to the position 3 amino group was also known, but this natural substance has no antimicrobial activity and was left as a substance of unknown activity. The present inventors discovered that this 3-N-leucyl-3-epi-5-dehydronegamycin also has potent read-through activity and perfected the pharmaceutical composition of the present invention.
One aspect of the present invention is a pharmaceutical composition containing a compound represented by the following general formula (3) or a salt or solvate of the compound.
Figure US09371274-20160621-C00028
in which R1, R2, R3, R4, R5, R6, R7, and n are as defined in general formula (1).
One aspect of the present invention is a pharmaceutical composition containing a compound represented by the following general formula (5) or a salt or solvate of the compound.
Figure US09371274-20160621-C00029
in which R1, R2, X, R3, R4, R5, R6, and R7 are as defined in general formula (1).
One aspect of the present invention is a pharmaceutical composition containing a compound wherein R1 and R2 in general formula (1), (3), or (5) are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded and this amino acid residue is represented by formula (2) below, or a salt or solvate of the compound.
Figure US09371274-20160621-C00030
in which
    • R8 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, where substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
    • R9, R10 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group,
    • R9 and R10 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • m is an integer of 0-4.
One aspect of the present invention is a pharmaceutical composition containing a compound wherein the N-linked amino acid residue of X in general formula (1) or (5) is represented by the following formula (4), or a salt or solvate of the compound.
Figure US09371274-20160621-C00031
in which
    • R11 is hydrogen or an optionally substituted C1-C6 alkyl group;
    • R12 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, where substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
    • R13, R14 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R8 and R9 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members (for example, oxazolidine), where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group;
    • p is an integer of 0-3.
Examples of substituents that the optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group of R12 in formula (4) can have are a halogen, hydroxyl group, carboxyl group, amino group, and amide group. Optionally substituted C1-C10 alkyl groups may be linear or branched and may form a cyclic structure.
A preferred embodiment of the present invention is a pharmaceutical composition containing a compound, wherein R12 is an optionally substituted C4-C9 alkyl group, or a salt or solvate of the compound.
A preferred embodiment of the present invention is a pharmaceutical composition containing a compound, wherein R12 is an optionally substituted C7-C9 alkyl group, or a salt or solvate of the compound.
The pharmaceutical composition of the present invention can be used in the treatment or prevention of nonsense mutation-type diseases. Nonsense mutation-type disease means a disease caused by the production of a stop codon midway in a gene (premature stop codon) due to a point mutation, deletion, insertion, or the like on a gene resulting in suppression of the expression of a normally functioning protein. Moreover, nonsense mutation-type disease means a disease caused by inhibition of the expression of a protein in association with degradation of mRNA containing a premature stop codon. Examples of nonsense mutation-type diseases include muscular dystrophy, Duchenne muscular dystrophy, infantile neuronal ceroid lipofuscinosis, multiple sclerosis, Alzheimer's disease, Tay-Sachs disease, neurodegenerative disease, Parkinson's disease, and other such central nervous diseases; rheumatoid arthritis, graft-versus-host disease, and other such autoimmune diseases; arthritis and other such inflammatory diseases; hemophilia, von Willebrand disease, ataxia telangiectasia, β-thalassemia, kidney stones, and other such blood diseases; osteogenesis imperfecta, liver cirrhosis, and other such collagen diseases; Other examples include neurofibromatosis, bullous disease, lysosomal storage disease, Hurler disease, familial hypercholesterolemia, cerebellar ataxia, nodular sclerosis, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial cholesterolemia, pigmentary retinopathy, amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, Niemann-Pick disease, Marfan syndrome, and the like. Cancer is also another example. For example, lung cancer, colorectal cancer, stomach cancer, esophageal cancer, kidney cancer, pancreatic cancer, liver cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, skin cancer, brain tumors, and the like are included as cancers in which a nonsense mutation of a p53 gene or another such suppressor gene participates. Among these, it can preferably be used in muscular dystrophy, cystic fibrosis, Hurler disease, and infantile neuronal ceroid lipofuscinosis. The pharmaceutical composition may contain two or more of the above compounds.
The compounds of the present invention can be used as a prodrug. Specifically, the water solubility of the compound decreases and the liposolubility increases when the part represented by —COOR7 is an ester in compounds of the above formulas (1), (3), and (5). Compounds of increased liposolubility promote systemic absorption and become an active form while being absorbed due to esterase present in the digestive tract and the like, increasing enteral absorption, or reach a site at which they are to act within the body and there they are converted into a carboxyl group by esterase present in the organs and tissues and can act at the site reached. As a result, the bioavailability is increased in the end. The compounds of the present invention also include compounds in the form of such prodrugs and also include compounds not in the form of prodrugs.
The route of administration of the pharmaceutical composition of the present invention is not limited, and it can be administered by oral administration, subcutaneous administration, intracutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, per nasal administration, intra-oral administration, transmucosal administration, and the like. The dosage form is also not limited. For example, tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, tinctures, syrups, extracts, and elixirs can be made for oral administration. As parenteral agents, for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and other such injections; percutaneous administration or adhesive skin patches, ointments, or lotions; sublingual agents or oral adhesive patches for intra-oral administration; and aerosols for per nasal administration can be made. For example, a pharmaceutical composition of the present invention can be administered as an injection (for example, an intramuscular injection for direct administration to a muscle) to treat muscular dystrophy. The pharmaceutical composition of the present invention may be in the form of a sustained-release or slow-release agent.
The pharmaceutical composition of the present invention can contain various pharmaceutically acceptable components. Examples include excipients, disintegrating agents, diluents, lubricants, flavorings, colorings, sweeteners, flavoring agents, suspending agents, wetting agents, emulsifiers, dispersing agents, auxiliary agents, preservatives, buffers, binders, stabilizers, coating agents, and the like. A plurality of these may be contained.
The dose of the pharmaceutical composition of the present invention is not limited, but is selected as is appropriate in accordance with the efficacy of the components contained, the dosage form, route of administration, type of disease, weight, age, medical conditions, and other such characteristics of the patient being administered, the judgment of a physician, and the like. For example, it is a range of approximately 0.01 μg to approximately 100 mg, preferably approximately 0.1 μg to approximately 1 mg, per kilogram of the patient's body weight. The dose can be administered from once to divided over several times per day or may be administered intermittently once every several days or every several weeks.
The read-through activity of compounds of the present invention can be tested, for example, by constructing a vector containing (i) a promoter, (ii) a first translation initiation codon and first reporter gene positioned downstream of the above promoter, (iii) a second translation initiation codon and second reporter gene positioned downstream of the above first reporter gene, (iv) a sequence containing a premature stop codon from a causative gene of a nonsense mutation-type disease positioned between the above first reporter gene and second translation initiation codon, and (v) a translation stop codon positioned downstream of the above second reporter gene, introducing this vector into host cells or creating transgenic animals with the vector introduced into animals, and using these host cells or transgenic animals. Specifically, a compound is administered to the host cells or transgenic animals, and the ratio of the expression level of the second reporter gene to the first reporter gene is compared between a case with and a case without addition of the test compound. The higher the expression level of the second reporter gene is, the higher the read-through activity can be judged to be. In this case, for example, a CMV promoter, β-actin promoter, or the like can be used as the promoter. A β-galactosidase gene, luciferase gene, GFP (green fluorescent protein) gene, CAT (chloramphenicol acetyl transferase) gene, or the like can be used as the first reporter gene and second reporter gene. A sequence containing a stop codon (TAA, TAG, or TGA) in a reading frame may be used as the sequence containing a premature stop codon from the causative gene of a nonsense-type disease. For example, a gene containing a premature stop codon of a dystrophin gene of mdx mice may be used.
EXAMPLES A. Synthesis of Compounds of the Present Invention Example 1 Synthesis of Example Compound 2 ((S)-2-(2-(3,6-diaminohexanoyl)-1-methylhydrazinyl)acetic acid)
Example compound 2 was synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00032
(1) Synthesis of Compound 1 ((S)-2,5-bis(tert-butoxycarbonylamino) pentylmethanesulfonate)
Figure US09371274-20160621-C00033
N-methylmorpholine (336 μL, 3.01 mmol) and isobutyl chloroformate (395 μL, 3.01 mmol) were added to a THF (3 mL) solution of Boc-Orn(Boc)-OH (1.00 g, 3.01 mol) at −15° C. and stirred for 10 minutes at the same temperature. The reaction solution was filtered, washed with THF, and a water (1.5 mL) solution of sodium borohydride (171 mg, 4.52 mmol) was added to the mother liquor obtained in an ice-salt bath and stirred for 20 minutes at the same temperature. After extraction by diethyl ether, the organic layer was washed with water and saturated saline and dried by Na2SO4. After filtration, a colorless oily substance (523 mg) was obtained by distilling off the mother liquor under reduced pressure. This was used in the next reaction without refining.
The residue obtained was then dissolved in dichloromethane (8 mL), and triethylamine (296 μL, 2.14 mmol) and methanesulfonyl chloride (190 μL, 2.46 mmol) were added while cooling by ice and stirred overnight at room temperature. Water was added to the reaction solution while cooling by ice, followed by extraction by chloroform. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white powder was obtained by refining the residue obtained by silica gel chromatography (chloroform: methanol=40:1). (381 mg, 0.319 mmol, three steps, 32%)
1HNMR (300 MHz, CDCl3) δ4.80-4.46 (m, 2H), 4.26 (dd, J=10 and 4.0 Hz, 1H), 4.18 (dd, J=10 and 4.1 Hz, 1H), 3.95-3.76 (m, 1H), 3.23-3.06 (m, 2H), 3.04 (s, 3H), 1.79-1.48 (m, 4H), 1.44 (s, 18H).
(2) Synthesis of Compound 2 ((S)-tert-butyl 5-cyanopentan-1,4-diyldicarbamate)
Figure US09371274-20160621-C00034
18-crown-6 (239 mg, 0.905 mmol) and potassium cyanide (157 mg, 2.41 mmol) were added to an acetonitrile (6 mL) solution of compound 1 (239 mg, 0.603 mmol) and heat refluxed overnight. After the reaction solution had returned to room temperature, saturated sodium bicarbonate aqueous solution was added while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=30:1). (146 mg, 0.446 mmol, 74%)
1HNMR (300 MHz, CDCl3) δ4.87 (brd, 1H), 4.68 (brs, 1H), 3.82 (brs, 1H), 3.23-3.07 (m, 2H), 2.74 (dd, J=17 and 5.4 Hz, 1H), 2.54 (dd, J=17 and 4.2 Hz, 1H), 1.75-1.53 (m, 4H), 1.45 (s, 18H).
(3) Synthesis of Compound 3 ((S)-tert-butyl 2-(2-(3,6-bis(tert-butoxycarbonylamino)hexanoyl)-1-methylhydrazinyl)acetate)
Figure US09371274-20160621-C00035
Potassium hydroxide (197 mg, 3.51 mmol) was added to an ethanol:water=2:1 (3 mL) solution of compound 2 (115 mg, 0.351 mmol) and stirred for 4 hours 45 minutes at 80° C. After the reaction solution had returned to room temperature, the solvent was distilled off under reduced pressure. 1 M hydrochloric acid was added while cooling by ice to bring the pH to 1-2, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, the residue obtained was dissolved in DMF (3 mL), and PTSA.H2N—N(Me)CH2CO2t-Bu (233 mg, 0.702 mmol) and HOBt.H2O (108 mg, 0.702 mmol) were added. Triethylamine (97.3 μL, 0.702 mmol) and EDC.HCl (135 mg, 0.702 mmol) were added sequentially while cooling by ice and stirred for two hours at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=80:1). (40.1 mg, 0.082 mmol, two steps, 23%)
1HNMR (300 MHz, CDCl3) δ7.90 (s) and 7.36 (brd, total 1H), 5.54-5.18 (m, 1H), 4.78-4.66 (m, 1H), 4.00-3.77 (m, 1H), 3.63-3.31 (m, 2H), 3.10-3.01 (m, 2H), 2.90-2.48 (m) and 2.41-2.20 (m, total 5H), 1.98-1.10 (m, 31H).
(4) Synthesis of Example Compound 2
Figure US09371274-20160621-C00036
4 M hydrochloric acid/dioxane (2 mL) was added to compound 3 (66.9 mg, 0.137 mmol) while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 0-5% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (44.9 mg, 97.5 μmol, 71%)
1HNMR (300 MHz, D2O) δ3.58-3.39 (m, 3H), 2.97-2.77 (m, 2H), 2.60-2.29 (m, 5H), 1.70-1.45 (m, 4H); HRMS(ES+) calcd for C9H21N4O3 (M++H) 233.1614. found 233.1614.
Example 2 Synthesis of Example Compound 5 ((S)-ethyl 2-(2-(3,6-diaminohexanoyl)-1-methylhydrazinyl)acetate)
Example compound 5 was synthesized by the following synthesis scheme.
Figure US09371274-20160621-C00037
(1) Synthesis of Compound 5 ((S)-5-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino)pentylmethanesulfonate)
Figure US09371274-20160621-C00038
N-methylmorpholine (1.97 mL, 15.0 mmol) and isobutyl chloroformate (1.68 mL, 15.0 mmol) were added to a THF (14 mL) solution of Boc-Orn(Cbz)-OH (5.00 g, 13.6 mmol) at −15° C. and stirred for 10 minutes at the same temperature. The reaction solution was filtered and washed with THF. A water (4 mL) solution of sodium borohydride (772 mg, 20.4 mmol) was added to the mother liquor obtained in an ice-salt bath and stirred for 10 minutes at the same temperature. Water was added to the reaction solution while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, a colorless oily substance was obtained by distilling off the mother liquor under reduced pressure. This was used in the next reaction without refining.
The residue obtained was then dissolved in dichloromethane (68 mL), and triethylamine (2.83 mL, 20.4 mmol) and methanesulfonyl chloride (3.14 mL, 40.8 mmol) were added while cooling by ice and stirred for 30 minutes at room temperature. Water was added to the reaction solution while cooling by ice, followed by extraction by chloroform. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white powder was obtained by refining the residue obtained by recrystallization (hexane: ethyl acetate). (4.49 g, 10.4 mmol, three steps, 77%)
[α]25D-17.2° (c0.34, CHCl3); 1HNMR (400 MHz, CDCl3) δ7.43-7.30 (m, 5H), 5.20-5.05 (s, 2H), 4.99-4.85 (brs, 1H), 4.80-4.62 (brd, 1H), 4.30-4.08 (M, 2H), 3.94-3.76 (brs, 1H), 3.31-3.14 (m, 2H), 3.02 (s, 3H), 1.79-1.49 (m, 4H), 1.44 (s, 9H); 13CNMR (100 MHz, CDCl3) δ156.5, 155.4, 136.5, 128.6, 128.2, 80.0, 71.0, 66.7, 49.5, 40.6, 37.3, 28.3, 26.4; HRMS(ES+) calcd for C19H30N2O7SNa (M++NA) 453.1671. found 453.1645.
(2) Synthesis of Compound 6 ((S)-benzyl tert-butyl(5-cyanopentan-1,4-diri)dicarbamate)
Figure US09371274-20160621-C00039
18-crown-6 (221 mg, 0.837 mmol) and potassium cyanide (136 mg, 2.09 mmol) were added to an acetonitrile (3.5 mL) solution of compound 5 (300 mg, 0.697 mmol) and heat refluxed for 30 minutes. After the reaction solution had returned to room temperature, saturated sodium bicarbonate aqueous solution was added while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (hexane:ethyl acetate=2:1). (206 mg, 0.570 mmol, 82%)
[α] 25D-41.2° (c0.74, CHCl3); 1HNMR (400 MHz, CDCl3) δ7.42-7.28 (m, 5H), 5.25-5.12 (m, 1H), 5.08 (s, 2H), 5.06-4.92 (brd, 1H), 3.92-3.69 (brs, 1H), 3.30-3.09 (m, 2H), 2.68 (dd, J=17 and 5.0 Hz, 1H), 2.47 (dd, J=17 and 3.7 Hz, 1H), 1.71-1.49 (m, 4H), 1.43 (s, 9H); 13CNMR (100 MHz, CDCl3) δ156.5, 155.1, 136.4, 128.5, 128.1, 128.0, 117.3, 80.0, 66.6, 47.0, 40.3, 30.5, 28.2, 26.4, 23.9; HRMS(ES+) calcd for C19H27N3O4Na (M++Na) 384.1899. found 384.1857.
(3) Synthesis of Compound 7 ((S)-6-(benzyloxycarbonylamino)-3-(tert-butoxycarbonylamino)hexanal)
Figure US09371274-20160621-C00040
Diisobutyl aluminum hydride (1.00 M toluene solution, 47.7 mL, 47.7 mmol) was added dropwise at −78° C. to an anhydrous dichloromethane (80 mL) solution of compound 6 (5.74 g, 15.9 mmol) under an argon atmosphere. The temperature of the reaction solution was raised to −50° C., and it was stirred for 1 hour 20 minutes. Methanol and saturated sodium potassium tartrate aqueous solution were added to the reaction solution at the same temperature, and the temperature was raised to room temperature, followed by extraction by chloroform. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless oily substance was obtained by refining the residue obtained by silica gel chromatography (hexane:ethyl acetate=1:1). (1.58 g, 5.52 mmol, 35%)
1HNMR (300 MHz, CDCl3) δ9.74 (s, 1H), 7.40-7.29 (m, 5H), 5.09 (s, 2H), 4.92-4.81 (m, 1H), 4.77-4.63 (m, 1H), 4.10-3.95 (m, 1H), 3.35-3.16 (m, 2H), 2.70-2.55 (m, 2H), 1.73-1.48 (m, 4H), 1.42 (s, 9H).
(4) Synthesis of Compound 8 ((S)-6-(benzyloxycarbonylamino)-3-(tert-butoxycarbonylamino)hexanoic acid)
Figure US09371274-20160621-C00041
2-Methyl-2-butene (2.30 mL, 21.6 mmol) and sodium dihydrogen phosphate (1.69 g, 10.8 mmol) were added to a t-butyl alcohol: water=2:1 (14 mL) solution of compound 7 (1.58 g, 4.33 mmol) and stirred for 10 minutes at room temperature. Sodium chlorite (1.57 g, 17.3 mmol) was then added and stirred for 10 minutes at room temperature. Saturated ammonium chloride aqueous solution was added to the reaction solution while cooling by ice, followed by extraction by chloroform. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless oily substance was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=50:1). (1.52 g, 4.00 mmol, 92%)
1HNMR (300 MHz, CDCl3) δ7.35-7.32 (m, 5H), 5.09-5.07 (m, 2H), 3.21-3.16 (m, 2H), 2.50-2.48 (m, 2H), 1.58-1.48 (m, 4H), 1.42 (s, 9H); HRMS(ES+) calcd for C19H29N2O6 (m++H) 381.2026. found 381.2020.
(5) Synthesis of Compound 9 ((S)-ethyl 2-(2-(6-benzyloxycarbonylamino)-3-(tert-butoxycarbonylamino)hexanoyl)-1-methylhydrazinyl)acetate)
Figure US09371274-20160621-C00042
H2N—N(Me)CH2CO2Et (56.4 mg, 0.427 mmol) and HOBt.H2O (65.4 mg, 0.427 mmol) were added to a DMF (2 mL) solution of compound 8 (81.1 mg, 0.213 mmol). Triethylamine (59.2 μL, 0.427 mmol) and EDC.HCl (81.8 mg, 0.427 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=80:1) (55.5 mg, 0.112 mmol, 53%)
[α]25D-15.5° (c0.38, CHCl3); 1HNMR (400 MHz, CDCl3) δ7.76 (s, 1H), 7.45-7.29 (m, 5H), 5.47-5.18 (m, 1H), 5.08 (s, 2H), 5.05-4.89 (m, 1H), 4.20 (q, J=7.1 Hz, 2H), 4.00-3.78 (m, 1H), 3.73-3.42 (m, 2H), 3.21 (t, J=6.0 Hz, 2H), 2.92-2.49 (m, 4H), 2.40-2.18 (m, 1H), 1.86-1.34 (m, 13H), 1.28 (t, J=7.2 Hz, 3H); 13CNMR (100 MHz, CDCl3) δ 170.8, 169.7, 156.5, 155.7, 136.7, 128.5, 128.08, 128.05, 79.2, 66.6, 60.9, 57.7, 47.6, 44.1, 40.7, 39.3, 32.0, 28.5, 26.7, 14.0; HRMS(ES+) calcd for C24H39N4O7 (M++H) 495.2819. found 495.2816.
(6) Synthesis of Example Compound 5
Figure US09371274-20160621-C00043
10% Pd/C (4.4 mg) was added to a methanol (1 mL) solution of compound 9 (44.0 mg, 89.0 μmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for 30 minutes at 50° C. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 4 M hydrochloric acid/dioxane (2 mL) was added to the residue while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 5-10% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (19.3 mg, 39.6 μmol, two steps, 44%)
1HNMR (300 MHz, D2O) δ 4.10 (q, J=6.9 Hz, 2H), 3.62-3.46 (m, 3H), 3.01-2.86 (m, 1H), 2.62-2.35 (m, 5H), 1.77-1.58 (m, 4H), 1.16 (t, J=6.6 Hz, 3H); HRMS(ES+) calcd for C11H25N4O3 (M++H) 261.1927. found 261.1926.
Example 3 Synthesis of Example Compound 9 ((S)-2-(2-(3,6-diaminohexanoyl)-1-ethylhydrazinyl)acetic acid) (1) Synthesis of Compound 11 ((S)-tert-butyl 2-(2-(6-(benzyloxycarbonylamino)-3-(tert-butoxycarbonylamino)hexanoyl-1-ethylhydrazinyl)acetate
Figure US09371274-20160621-C00044
PTSA.H2N—N(Et)CH2CO2t-Bu (167 mg, 0.482 mmol) and HOBt.H2O (73.9 mg, 0.482 mmol) were added to a DMF (3 mL) solution of compound 8 (91.7 mg, 0.241 mmol) synthesized in Example 2. Triethylamine (66.8 μL, 0.482 mmol) and EDC.HCl (92.4 mg, 0.482 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=60:1). (71.7 mg, 0.134 mmol, 55%)
[α]25D-6.31° (c0.52, CHCl3); 1HNMR (400 MHz, CDCl2) δ 7.73 (s) and 7.15 (d, J=26 Hz, total 1H), 7.42-7.28 (m, 5H), 5.59-5.24 (m, 1H), 5.09 (s, 2H), 5.05-4.91 (brs, 1H), 3.98-3.78 (m, 1H), 3.69-3.38 (m, 2H), 3.30-3.13 (m, 2H), 3.07-2.78 (m, 2H), 2.74-2.21 (m, 2H), 1.70-1.34 (m, 22H), 1.08 (t, J=7.1 Hz, 3H); 13CNMR (100 MHz, CDCl3) δ 170.5, 169.4, 156.5, 155.7, 136.7, 128.4, 128.06, 128.03, 82.4, 79.3, 66.5, 57.3, 50.4, 47.5, 40.8, 36.4, 32.0, 28.4, 28.2, 26.6, 12.7; HRMS(ES+) calcd for C27H45N4O7 (M++H) 537.3288. found 537.3303.
(2) Synthesis of Example Compound 9
Figure US09371274-20160621-C00045
10% Pd/C (5.5 mg) was added to a methanol (2 mL) solution of compound 11 (55.8 mg, 0.104 mmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for 30 minutes at 50° C. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 4 M hydrochloric acid/dioxane (2 mL) was added to the residue while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure and the residue obtained was dissolved in water. A colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 10-20% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (26.7 mg, 56.2 μmol, two steps, 54%)
1HNMR (300 MHz, CDCl3) δ 3.65-3.52 (m, 3H), 3.03-2.80 (m, 4H), 2.55 (dd, J=17 and 5.4 Hz, 2H), 1.74-1.57 (m, 4H), 0.97 (t, J=7.2 Hz, 3H); HRMS(ES+) calcd for C10H23N4O3 (M++H) 247.1770. found 247.1777.
Example 4 Synthesis of Example Compound 7 (2-(2-((S)-6-amino-3-((S)-2-amino-4-methylpentanamide)hexanoyl)-1-methylhydrazinyl)acetic acid)
Example compound 7 was synthesized by the following synthesis scheme.
Figure US09371274-20160621-C00046
(1) Synthesis of Compound 13 ((S)-benzyl 2-(2-(6-(benzyloxycarbonylamino)-3-(tert-butoxycarbonylamino)hexanoyl)-1-methylhydrazinyl)acetate)
Figure US09371274-20160621-C00047
H2N—N(Me)CH2CO2Bn (163 mg, 0.842 mmol) and HOBt.H2O (129 mg, 0.842 mmol) were added to a DMF (2 mL) solution of compound 8 (160 mg, 0.421 mmol). Triethylamine (117 μL, 0.842 mmol) and EDC.HCl (161 mg, 0.842 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=80:1). (139 mg, 0.250 mmol, 59%)
[α]25D-6.49° (c0.75, CHCl3); 1HNMR (400 MHz, CDCl2) δ 7.75 (s) and 7.42-7.19 (m, total, 11H), 5.48-4.76 (m, 6H), 4.00-3.77 (m, 1H), 3.77-3.45 (m, 2H), 3.30-3.05 (m, 2H), 2.90-2.48 (m, 4H), 2.40-2.10 (m, 1H), 1.92-1.46 (m, 4H), 1.40 (s, 9H); 13CNMR (100 MHz, CDCl2) δ 170.6, 169.3, 156.5, 155.6, 136.7, 135.1, 128.71, 128.67, 128.62, 128.51, 128.43, 128.06, 79.2, 66.6, 58.9, 57.7, 47.5, 44.1, 40.7, 39.3, 32.0, 28.4, 26.6; HRMS(ES+) calcd for C29H41N4O7 (M++H) 557.2975. found 557.2985.
(2) Synthesis of Compound 14 ((7S,10S)-benzyl 7-(3-(benzyloxycarbonylamino)propyl)-10-isobutyl-3,14,14-trimethyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate)
Figure US09371274-20160621-C00048
4 M hydrochloric acid/dioxane (3 mL) was added to compound 13 (66.2 mg, 0.119 mmol) while cooling by ice and stirred for 30 minutes at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was used in the next reaction without refining. The residue was dissolved in DMF (2 mL), and Boc-Leu-OH (35.6 mg, 0.143 mmol) and HOBt.H2O (20.0 mg, 0.131 mmol) were added. Triethylamine (18.2 μL, 0.131 mmol) and EDCHCl (27.4 mg, 0.131 mmol) were added sequentially and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=80:1). (54.0 mg, 0.081 mmol, two steps 68%)
[α]25D-21.5° (c0.67, CHCl3); 1HNMR (400 MHz, CDCl3) δ 7.79 (s) and 7.45-7.29 (m, total 11H), 7.03 (brs, 1H), 5.25-5.00 (m, 5H), 4.94 (brs, 1H), 4.35-4.10 (m, 1H), 4.10-3.93 (brs, 1H), 3.77-3.46 (m, 2H), 3.28-3.08 (m, 2H), 2.93-2.43 (m, 4H), 2.37-2.16 (m, 1H), 1.98-1.20 (m, 16H), 1.03-0.75 (m, 6H); 13CNMR (100 MHz, CDCl3) δ 172.4, 170.6, 169.0, 156.58, 156.54, 136.72, 135.16, 128.73, 128.66, 128.52, 128.48, 128.45, 128.04, 80.0, 66.7, 66.6, 57.8, 53.6, 46.1, 44.2, 41.4, 40.7, 40.6, 38.8, 31.4, 28.3, 26.4, 23.9, 22.0; HRMS(ES+) calcd for C35H52N5O8 (M++H) 670.3816. found 670.3808.
(3) Synthesis of Example Compound 7
Figure US09371274-20160621-C00049
10% Pd/C (3.1 mg) was added to a methanol (2 mL) solution of compound 14 (31.3 mg, 46.8 μmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for 45 minutes at 50° C. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 4 M hydrochloric acid/dioxane (2 mL) was added to the residue while cooling by ice and stirred for 10 minutes at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A white, cottony substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 10-15% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (16.1 mg, 28.0 μmol, two steps, 60%)
1HNMR (300 MHz, D2O) δ 4.23-4.00 (m, 1H), 3.92-3.74 (m, 1H), 3.50 (s, 2H), 2.89 (t, J=6.3 Hz, 2H), 2.56 (s, 3H), 2.36 (dd, J=15 and 5.4 Hz, 1H), 2.21 (dd, J=15 and 9 Hz, 1H), 1.72-1.35 (m, 7H), 0.83 (dd, J=4.2 Hz, 6H); HRMS(ES+) calcd for C15H32N5O4 (M++H) 346.2454. found 346.2452.
Example 5 Synthesis of Example Compound 3 ((S)-2-(2-(3,5-diaminopentanoyl)-1-methylhydrazinyl)acetic acid)
Example compound 3 was synthesized by the following synthesis scheme.
Figure US09371274-20160621-C00050
(1) Synthesis of Compound 16 ((S)-2,4-bis(tert-butoxycarbonylamino) butylmethanesulfonate)
Water (36 mL) was added to a THF (145 mL) solution of Boc-Gln-OH (15.0 g, 60.9 mmol), and iodobenzene diacetate (23.5 g, 73.1 mmol) was added while cooling by ice and stirred for 4 hours 30 minutes at room temperature. The reaction solution was distilled under reduced pressure, and water was added to the residue obtained, followed by extraction by ethyl acetate. The water layer was distilled off under reduced pressure, and the residue obtained was washed multiple times with cold chloroform, and a white powder (10.0 g) was obtained by drying. This was used in the next reaction without refining.
Sodium hydroxide (2.02 g, 50.4 mmol) and (Boc)2O (12.0 g, 55.0 mmol) were added while cooling by ice to a dioxane: water=2:1 (225 mL) solution of the above powder (10.0 g, 45.8 mmol) and stirred for one hour at room temperature. The reaction solution was distilled off under reduced pressure, and 1 M hydrochloric acid was added while cooling by ice to the residue obtained to bring the pH to 1-2, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, a yellow oily substance was obtained by distilling off the mother liquor under reduced pressure. This was used in the next reaction without refining.
The residue was dissolved in THF (90 mL), and N-methylmorpholine (5.63 mL, 50.4 mmol) and isobutyl chloroformate (6.62 mL, 50.4 mmol) were added at −15° C. and stirred for five minutes at the same temperature. The reaction solution was filtered, washed with THF, and a water (15 mL) solution of sodium borohydride (2.60 g, 68.7 mmol) was added in an ice-salt bath to the mother liquor obtained and stirred for five minutes at the same temperature. 1 M hydrochloric acid was added to the reaction solution while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, a colorless oily substance was obtained by distilling off the mother liquor under reduced pressure. This was used in the next reaction without refining.
Triethylamine (9.52 mL, 68.7 mmol) and methanesulfonyl chloride (10.6 mL, 137 mmol) were added to a dichloromethane (230 mL) solution of the residue and stirred overnight at room temperature. Water was added to the reaction solution while cooling by ice, followed by extracted by chloroform. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a colorless oily substance was obtained by refining the residue obtained by silica gel chromatography (hexane:ethyl acetate=2:1). (7.97 g, 20.9 mmol, four steps, 46%)
[α]25D-38.1° (c1.97, CHCl3); 1HNMR (400 MHz, CDCl3) δ 5.21-5.03 (brs, 1H), 5.03-4.78 (brd, 1H), 4.30 (dd, J=10 and 3.5 Hz, 1H), 4.23 (dd, J=10 and 4.4 Hz, 1H), 4.01-3.88 (m, 1H), 3.49-3.27 (brs, 1H), 3.12-2.94 (m, 4H), 1.87-1.55 (m, 2H), 1.44 (s, 18H); 13CNMR (100 MHz, CDCl3) δ 156.0, 155.7, 80.1, 79.4, 71.4, 47.3, 37.3, 36.9, 31.9, 28.4, 28.3; HRMS(ES+) calcd for C15H30N2O7SNa (M++Na) 405.1671. found 405.1671.
(2) Synthesis of Compound 17 ((S)-tert-butyl 4-cyanobutane-1,3-diyldicarbamate)
Figure US09371274-20160621-C00051
18-Crown-6 (2.76 g, 10.4 mmol) and potassium cyanide (2.72 g, 41.7 mmol) were added to an acetonitrile (105 mL) solution of compound 16 (7.97 g, 20.9 mmol) and stirred overnight at 40° C. After the reaction solution had returned to room temperature, saturated sodium bicarbonate aqueous solution was added while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (hexane:ethyl acetate=2:1). (4.08 g, 13.0 mmol, 62%)
[α]25D-64.1° (c1.14, CHCl3); 1HNMR (400 MHz, CDCl3) δ 5.03-4.72 (m, 2H), 4.00-3.83 (m, 1H), 3.48-3.26 (m, 1H), 3.16-2.97 (m, 1H), 2.74 (dd, J=17 and 5.4 Hz, 1H), 2.62 (dd, J=17 and 4.6 Hz, 1H), 1.90-1.67 (m, 2H), 1.45 (s, 9H), 1.44 (s, 9H); 13CNMR (100 MHz, CDCl3) δ 156.0, 155.3, 117.2, 80.4, 79.6, 45.0, 36.9, 34.3, 28.4, 28.3, 23.9; HRMS(ES+) calcd for C15H27N3O4Na (M++Na) 336.1899. found 336.1896.
(3) Synthesis of Compound 18 ((S)-tert-butyl 2-(2-(3,5-bis(tert-butoxycarbonylamino)pentanoyl)-1-methylhydrazinyl)acetate)
Figure US09371274-20160621-C00052
Potassium hydroxide (162 mg, 2.88 mmol) was added to an ethanol:water=2:1 (3 mL) solution of compound 17 (90.2 mg, 0.288 mmol) and stirred for 7 hours 30 minutes at 80° C. After the reaction solution had returned to room temperature, the solvent was distilled off under reduced pressure. 1 M hydrochloric acid was added while cooling by ice to bring the pH to 1-2, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure. The residue obtained was dissolved in DMF (2 mL), and PTSA.H2N—N(Me)CH2CO2t-Bu (191 mg, 0.576 mmol) and HOBt.H2O (88.2 mg, 0.576 mmol) were added. Triethylamine (79.8 μL, 0.576 mmol) and EDC.HCl (110 mg, 0.576 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature.
The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=100:1). (51.3 mg, 0.108 mmol, two steps 38%)
[α]25D-40.1° (c0.52, CHCl3); 1HNMR (400 MHz, CDCl3) δ 7.94 (s) and 7.38 (brd, total 1H), 5.83-5.49 (m, 1H), 5.49-5.11 (m, 1H), 4.15-3.80 (m, 1H), 3.69-3.46 (m, 2H), 3.46-3.20 (m, 1H), 3.05-2.81 (m, 1H), 2.81-2.48 (m, 4H), 2.48-2.18 (m, 1H), 1.75-1.18 (m, 29H); 13CNMR (100 MHz, CDCl3) δ 174.7, 170.2, 169.1, 156.1, 82.4, 79.3, 79.0, 58.2, 45.1, 43.9, 38.9, 37.1, 35.2, 28.44, 28.36, 28.2; HRMS(ES+) calcd for C22H42N4O7Na (M++Na) 497.2951. found 497.2959.
(4) Synthesis of Example Compound 3
Figure US09371274-20160621-C00053
4 M hydrochloric acid/dioxane (2 mL) was added while cooling by ice to compound 18 (29.3 mg, 61.9 μmol) and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A colorless oily substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 0-5% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (14.6 mg, 35.3 μmol, 57%)
1HNMR (300 MHz, D2O) δ 3.60-3.50 (m, 1H), 3.47 (s, 2H), 2.93 (t, J=8.8 Hz, 2H), 2.51-2.34 (m, 5H), 1.99-1.79 (m, 2H); HRMS(ES+) calcd for C8H19N4O3 (M++H) 219.1457. found 219.1455.
Example 6 Synthesis of Example Compounds 14-18
Example compounds 14-18 were synthesized by the following synthesis scheme. Furthermore, in the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00054
(A) Compounds 20a-20e (1) Synthesis of Compound 20a
Figure US09371274-20160621-C00055
Compound 20a was synthesized by the same method as compound 14 using compound 13 (82.9 mg, 0.149 mmol) (white solid, 92.5 mg, 0.132 mmol, two steps 88%).
HRMS(ES+) calcd for C38H50N5O8 (M++H) 704.3659 found 704.3652.
(2) Synthesis of Compound 20b
Figure US09371274-20160621-C00056
Compound 20b was synthesized by the same method as compound 14 using compound 13 (61.2 mg, 0.110 mmol) (colorless solid, 39.3 mg, 54.6 μmol, two steps 50%).
HRMS(ES+) calcd for C38H50N5O9 (M++H) 720.3609. found 720.3595.
(3) Synthesis of Compound 20c
Figure US09371274-20160621-C00057
Compound 20c was synthesized by the same method as compound 14 using 13 (71.7 mg, 0.129 mmol) (white solid, 77.8 mg, 0.119 mmol, two steps, 92%).
HRMS(ES+) calcd for C34H49N5O8Na (M++Na) 678.3479. found 678.3481.
(4) Synthesis of Compound 20d
Figure US09371274-20160621-C00058
Compound 20d was synthesized by the same method as compound 14 using compound 13 (52.0 mg, 93.5 μmol) (colorless solid, 51.7 mg, 84.3 μmol, two steps 90%).
HRMS(ES+) calcd for C31H44N5O8 (M++H) 614.3190. found 614.3175.
(5) Synthesis of Compound 20e
Figure US09371274-20160621-C00059
Compound 20e was synthesized by the same method as compound 14 using compound 13 (79.7 mg, 0.143 mmol) (white solid, 90.8 mg, 0.136 mmol, two steps 95%).
HRMS(ES+) calcd for C35H51N5O8Na (M++Na) 692.3635. found 692.3638.
(B) Example Compounds 14-18 (1) Synthesis of Example Compound 14
Figure US09371274-20160621-C00060
Synthesized by the same method as example compound 7 using compound 20a (43.7 mg, 62.1 μmol) (colorless solid, 11.2 mg, 18.4 μmol, two steps, 30%).
HRMS(ES+) calcd for C18H30N5O4 (M++H) 380.2298. found 380.2290.
(2) Synthesis of Example Compound 15
Figure US09371274-20160621-C00061
Synthesized by the same method as example compound 7 using compound 20b (27.8 mg, 38.6 μmol) (colorless solid, 8.25 mg, 13.2 μmol, two steps, 34%).
HRMS(ES+) calcd for C18H30N5O5 (M++H) 396.2247. found 396.2258.
(3) Synthesis of Example Compound 16
Figure US09371274-20160621-C00062
Synthesized by the same method as example compound 7 using compound 20c (40.4 mg, 61.6 μmol) (colorless solid, 11.2 mg, 20.0 μmol, two steps, 33%).
HRMS(ES+) calcd for C14H30N5O4 (M++H) 332.2298. found 332.2286.
(4) Synthesis of Example Compound 17
Figure US09371274-20160621-C00063
Synthesized by the same method as example compound 7 using compound 20d (30.8 mg, 50.2 μmol) (colorless solid, 12.3 mg, 23.8 μmol, two steps, 47%).
HRMS(ES+) calcd for C11H24N5O4 (M++H) 290.1828. found 290.1829.
(5) Synthesis of Example Compound 18
Figure US09371274-20160621-C00064
Synthesized by the same method as example compound 7 using compound 20e (43.1 mg, 64.4 μmol) (colorless solid, 17.2 mg, 30.0 μmol, two steps, 47%).
HRMS(ES+) calcd for C15H32N5O4 (M++H) 346.2454. found 346.2459.
Example 7 Synthesis of Example Compounds 19-20
Example compounds 19-20 were synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00065
(A) Compounds 22a-22b (1) Synthesis of Compound 22a
Figure US09371274-20160621-C00066
Potassium hydroxide (1.85 g, 32.9 mmol) was added to an ethanol:water=2:1 (16 mL) solution of compound 17 (1.03 g, 3.29 mmol) and stirred for five hours at 80° C. After the reaction solution had returned to room temperature, the solvent was distilled off under reduced pressure. 1 M hydrochloric acid was added while cooling by ice to bring the pH to 1-2, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and the residue obtained was dissolved in DMF (16 mL), and H2N—N(Me)CH2CO2Bn (1.28 g, 6.58 mmol) and HOBt.H2O (1.03 g, 6.58 mmol) were added. Triethylamine (912 μL, 6.58 mmol) and EDC.HCl (1.26 g, 6.58 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid (771 mg, 1.52 mmol, two steps, 46%) was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=100:1).
HRMS(ES+) calcd for C25H41N4O7 (M++H) 509.2975. found 509.2981.
(2) Synthesis of Compound 22b
Figure US09371274-20160621-C00067
Compound 22b was synthesized by the same method as compound 22a using compound 17 (111 mg, 0.354 mmol) and H2N—N(Me)CH2CO2Bn(o-Br) (62.6 mg, 0.229 mmol) (white solid, 75.4 mg, 0.128 mmol, two steps, 36%).
HRMS(ES+) calcd for C25H40N4O7Br (M++H) 587.2080. found 587.2092.
(B) Example Compounds 19-20 (1) Synthesis of Example Compound 19
Figure US09371274-20160621-C00068
4 M hydrochloric acid/dioxane (2 mL) was added to compound 22a (64.9 mg, 0.128 mmol) while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A colorless solid (34.6 mg, 64.5 μmol, 50%) was obtained by refining by high-performance liquid chromatography (solvent: H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 15-45% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at UV 222 nm).
1H NMR (300 MHz, D2O) δ7.42-7.40 (m, 5H), 5.20 (s, 2H), 3.67-3.62 (m, 3H), 3.06 (t, J=8.22 Hz, 2H), 2.63 (s, 3H), 2.52-2.48 (m, 2H), 2.07-1.96 (m, 2H); HRMS(ES+) calcd for C15H25N4O3 (M++H) 309.1927. found 309.1915.
(2) Synthesis of Example Compound 20
Figure US09371274-20160621-C00069
4 M hydrochloric acid/dioxane (2 mL) was added to compound 22b (52.0 mg, 88.5 μmol) while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A white powder (26.9 mg, 43.9 μmol, 50%) was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 30-50% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at UV 222 nm).
HRMS(ES+) calcd for C15H24N4O3Br (M++H) 387.1032. found 387.1033.
Example 8 Synthesis of Example Compounds 21-24
Example compounds 21-24 were synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00070
(A) Compounds 24a-24d (1) Synthesis of Compound 24a
Figure US09371274-20160621-C00071
10% Pd/C (10.8 mg) was added to a methanol (2 mL) solution of compound 22a (108 mg, 0.212 mmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for 1 hour 30 minutes at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 3-Bromobenzyl alcohol (47.5 mg, 0.254 mmol) was added at room temperature to a DMF (2 mL) solution of the residue, and DMAP (2.59 mg, 21.2 μmol) and DCC (48.1 mg, 0.254 mmol) were added while cooling by ice and stirring, and stirred overnight at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was diluted by chloroform and filtered. The organic layer was washed with water and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid (84.6 mg, 0.144 mmol, two steps, 68%) was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=100:1).
HRMS(ES+) calcd for C25H40N4O7Br (M++H) 587.2080. found 587.2080.
(2) Synthesis of Compound 24b
Figure US09371274-20160621-C00072
Compound 24b was synthesized by the same method as compound 24a using compound 22a (120 mg, 0.236 mmol) and 4-bromobenzyl alcohol (53.0 mg, 0.283 mmol) (white solid, 71.6 mg, 0.122 mmol, two steps, 52%).
HRMS(ES+) calcd for C25H40N4O7Br (M++H) 587.2080. found 587.2078.
(3) Synthesis of Compound 24c
Figure US09371274-20160621-C00073
Compound 24c was synthesized by the same method as compound 24a using compound 22a (99.6 mg, 0.196 mmol) and 2-nitrobenzyl alcohol (36.0 mg, 0.235 mmol) (white solid, 28.3 mg, 51.1 μmol, two steps, 26%).
1H NMR (300 MHz, CDCl3) δ8.13 (d, J=8.02 Hz, 1H), 7.75-7.65 (m, 2H), 7.65-7.51 (m, 2H), 6.74-6.50 (m, 3H), 5.40-5.18 (brd, 1H), 4.08-3.86 (m, 1H), 3.86-3.59 (m, 2H), 3.48-3.25 (m, 1H), 3.03-2.84 (m, 1H), 2.84-2.49 (m, 4H), 2.49-2.19 (m, 1H), 1.77-1.58 (m, 2H), 1.43 (s, 18H).
(4) Synthesis of Compound 24d
Figure US09371274-20160621-C00074
Synthesized by the same method as compound 24a using compound 22a (114 mg, 0.224 mmol) and 2-methoxybenzyl alcohol (37.1 mg, 0.269 mmol) (white solid, 59.6 mg, 0.111 mmol, two steps, 49%).
HRMS(ES+) calcd for C26H43N4O8 (M++H) 539.3081. found 539.3079.
(B) Example Compounds 21-24 (1) Synthesis of Example Compound 21
Figure US09371274-20160621-C00075
Synthesized by the same method as example compound 20 using compound 24a (44.9 mg, 76.4 μmol) (white solid, 10.7 mg, 17.3 μmol, 23%).
1H NMR (400 MHz, D2O) δ7.64-7.58 (m, 2H), 7.43-7.33 (m, 2H), 5.20 (s, 2H), 3.78-3.67 (m, 3H), 3.11 (t, J=8.34 Hz, 2H), 2.67 (s, 3H), 2.60-2.49 (m, 2H), 2.16-1.96 (m, 2H); 13C NMR (100 MHz, D2O) δ170.60, 168.82, 137.73, 131.49, 130.94, 130.53, 127.03, 121.94, 66.2, 58.4, 46.0, 44.3, 35.6, 34.5, 29.8.
(2) Synthesis of Example Compound 22
Figure US09371274-20160621-C00076
Synthesized by the same method as example compound 20 using compound 24b (37.2 mg, 63.3 μmol) (white solid, 18.4 mg, 29.9 μmol, 47%).
HRMS(ES+) calcd for C15H24N4O3Br (M++H) 387.1032. found 387.1024.
(3) Synthesis of Example Compound 23
Figure US09371274-20160621-C00077
Synthesized by the same method as example compound 20 using compound 24c (28.3 mg, 51.1 μmol) (white solid, 21.8 mg, 37.5 μmol, 73%).
HRMS(ES+) calcd for C15H24N5O5 (M++H) 354.1777. found 354.1782.
(4) Synthesis of Example Compound 24
Figure US09371274-20160621-C00078
Synthesized by the same method as example compound 20 using compound 24d (30.4 mg, 56.5 μmol) (white solid, 20.8 mg, 36.8 μmol, 65%).
HRMS(ES+) calcd for C16H27N4O4 (M++H) 339.2032. found 339.2025.
Example 9 Synthesis of example compounds 25-28
Example compounds 25-28 were synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00079
(A) Synthesis of Compound 27
Figure US09371274-20160621-C00080
Ammonium acetate (23.1 g, 300 mmol) and molecular sieve 3A (24 g) were added to a methanol solution (285 mL) of dimethyl-1,3-acetonedicarboxylate (14.4 mL 100 mmol) and stirred for 5 hours 30 minutes at room temperature. Sodium cyanoborohydride (7.86 g, 125 mmol) was added, methanol-hydrochloric acid solution was added to pH 3, and the solution was stirred for 20 minutes at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was dissolved in water (200 mL) and washed with diethyl ether. Sodium carbonate was added to bring the water layer near pH 10 while cooling by ice and stirring. Di-tert-butoxydicarbonate (21.8 g, 100 mmol) was added at the same temperature and stirred overnight at room temperature. The reaction solution was extracted by diethyl ether, and after washing the extract with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (23.3 g, 84.6 mmol, 85%) was obtained by refining the residue obtained by silica gel column chromatography (hexane:ethyl acetate=4:1).
1H NMR (300 MHz, CDCl3) δ5.39-5.30 (brd, 1H), 4.38-4.28 (m, 1H), 3.69 (s, 6H), 2.74-2.59 (m, 4H), 1.43 (s, 9H).
(B) Synthesis of Compound 28
Figure US09371274-20160621-C00081
Acetone (4 mL) and esterase from porcine liver (Aldrich, 70.6 mg, 1200 units) were added to a phosphate buffer solution (pH 8, 0.5 M, 100 mL) of compound 27 (1.00 g, 3.60 mmol), maintained at pH 8 at 25° C., and stirred overnight. Concentrated hydrochloric acid was added to the reaction solution to bring the pH close to 1, and the solution was filtered. After extracting by chloroform and washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. Borane-tetrahydrofuran complex (1.0 M, 5.42 mL) was added dropwise while stirring at −78° C. to an anhydrous tetrahydrofuran solution (11 mL) of the residue under an argon atmosphere and stirred overnight at room temperature. Saturated ammonium chloride aqueous solution was added to the reaction solution while cooling by ice and stirring, followed by extraction by diethyl ether. After washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (588 mg, 2.37 mmol, 66%) was obtained by refining the residue obtained by silica gel column chromatography (hexane:ethyl acetate=4:1).
[α]25D-20.7° (c1.00, CHCl3), lit[α]25D-23.9° (c1.00, CHCl3); 1H NMR (300 MHz, CDCl3) δ5.42-5.36 (brs, 1H), 4.21-4.11 (m, 1H), 3.70 (s, 3H), 3.65-3.58 (m, 2H), 2.69-2.50 (m, 2H), 1.83-1.49 (m, 2H), 1.49 (s, 9H).
(C) Synthesis of Compound 29
Figure US09371274-20160621-C00082
Triethylamine (42.1 μL, 0.303 mmol) and methanesulfonyl chloride (31.1 μL, 0.404 mmol) were added while cooling by ice and stirring to a dichloromethane solution (1 mL) of compound 28 (50.0 mg, 0.202 mmol) and stirred for 2 hours minutes at room temperature. Water was added to the reaction solution while cooling by ice and stirring, followed by extraction by chloroform. After washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A mesilate (59.7 mg, 0.183 mmol, 90%), which was a white solid, was obtained by refining the residue obtained by silica gel column chromatography (hexane:ethyl acetate=1:1).
1H NMR (300 MHz, CDCl3) δ5.20-5.10 (brd, 1H), 4.35-4.22 (m, 2H), 4.19-4.01 (m, 1H), 3.70 (s, 3H), 3.04 (s, 3H), 2.68-2.53 (m, 2H), 2.05-1.96 (m, 2H), 1.44 (s, 9H).
Sodium azide (219 mg, 3.36 mmol) was added to a DMF (10 mL) of the above mesilate (365 mg, 1.12 mmol) and stirred overnight at 50° C. After the reaction solution had returned to room temperature, it was distilled under reduced pressure and a colorless oily substance (278 mg, 1.02 mmol, 91%) was obtained by refining the residue obtained by silica gel column chromatography (hexane:ethyl acetate=3:1).
1H NMR (300 MHz, CDCl3) δ5.13-5.03 (brd, 1H), 4.10-3.92 (m, 1H), 3.70 (s, 3H), 3.45-3.30 (m, 2H), 2.64-2.50 (m, 2H), 1.83-1.69 (m, 2H), 1.44 (s, 9H).
(D) Synthesis of Compound 30
Figure US09371274-20160621-C00083
Potassium hydroxide (310 mg, 5.52 mmol) was added while cooling by ice and stirring to a methanol-water (2:1, 9 mL) mixed solution of compound 29 (500 mg, 1.84 mmol) and stirred for 1 hour 30 minutes at room temperature. The reaction solution was distilled off under reduced pressure, and 1 M hydrochloric acid solution was added to bring the residue obtained close to pH 1 while cooling by ice and stirring, followed by extraction by ethyl acetate. After washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. PTSA.H2N—N(Me)CH2CO2t-Bu (1.22 g, 3.68 mmol) and HOBt.H2O (564 mg, 3.68 mmol) were added to a DMF solution (9 mL) of the residue, triethylamine (510 μL, 3.68 mmol) and EDC.HCl (705 mg, 3.68 mmol) were added while cooling by ice and stirring, and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. After washing the organic layer sequentially with saturated sodium bicarbonate aqueous solution, water, and saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (241 mg, 0.601 mmol, two steps 33%) was obtained by refining the residue obtained by silica gel column chromatography (chloroform:methanol=80:1).
HRMS(ES+) calcd for C17H33N6O5 (M++H) 401.2512. found 401.2509.
(E) Synthesis of Compound 31
Figure US09371274-20160621-C00084
10% Pd/C (13.5 mg) was stirred for 1 hour 30 minutes under a hydrogen atmosphere in a methanol solution (2 mL) of compound 30 (135 mg, 0.337 mmol). The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. Triethylamine (56.0 μL, 0.404 mmol) and nosyl chloride (82.2 mg, 0.371 mmol) were added to a dichloromethane solution (2 mL) and stirred for 35 minutes at room temperature. Water was added to the reaction solution while cooling by ice and stirring, followed by extraction by chloroform. After washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (164 mg, 0.293 mmol, two steps, 87%) was obtained by refining the residue obtained by silica gel column chromatography (chloroform:methanol=100:1).
HRMS(ES+) calcd for C23H38N5O8S (M++H) 560.2390. found 560.2386.
(F) Compounds 32a-32d (1) Synthesis of Compound 32a
Figure US09371274-20160621-C00085
Potassium carbonate (14.7 mg, 0.107 mmol) and 3-bromopropyne (7.28 μL, 97.8 μmol) were added at room temperature to a DMF solution (2 mL) of compound 31 (49.7 mg, 88.9 μmol) and stirred for 1 hour 45 minutes at 50° C. After the reaction solution had returned to room temperature, water was added and it was extracted by ethyl acetate. After washing the organic layer with saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (50.1 mg, 83.9 μmol, 94%) was obtained by refining the residue obtained by silica gel column chromatography (chloroform: methanol=100:1).
HRMS(ES+) calcd for C26H40N5O9S (M++H) 598.2547. found 598.2543.
(2) Synthesis of Compound 32b
Figure US09371274-20160621-C00086
Synthesized by the same method as compound 32a using compound 31 (61.5 mg, 0.110 mmol) and 1-bromopropane (55.0 μL, 0.550 mmol) (yellow oily substance, 60.3 mg, 0.100 mmol, 91%).
HRMS(ES+) calcd for C26H44N5O9S (M++H) 602.2860. found 602.2845.
(3) Synthesis of Compound 32c
Figure US09371274-20160621-C00087
Synthesized by the same method as compound 32a using compound 31 (60.5 mg, 0.108 mmol) and 1-bromopentane (66.9 μL, 0.540 mmol) (colorless oily substance, 62.7 mg, 99.6 μmol, 92%).
HRMS(ES+) calcd for C28H48N5O9S (M++H) 630.3173. found 630.3174.
(4) Synthesis of Compound 32d
Figure US09371274-20160621-C00088
Synthesized by the same method as compound 32a using compound 31 (58.2 mg, 0.104 mmol) and benzyl bromide (12.4 μL, 0.114 mmol) (colorless oily substance, 52.7 mg, 81.2 μmol, 78%).
HRMS(ES+) calcd for C30H44N5O9S (M++H) 650.2860. found 650.2842.
(G) Example Compounds 25-28 (1) Synthesis of Example Compound 25
Figure US09371274-20160621-C00089
Potassium carbonate (41.5 mg, 0.300 mmol) and thiophenol (30.6 μL, 0.300 mmol) were added to an acetonitrile solution (2 mL) of compound 32a (35.8 mg, 59.9 μmol) and stirred for 2 hours 15 minutes at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. 4 M hydrochloric acid/dioxane (2 mL) was added while cooling by ice and stirring to the residue obtained and stirred for one hour at room temperature. After distilling off the reaction solution, the residue was dissolved in water and refined by HPLC. A white solid (13.3 mg, 27.4 μmol, two steps, 46%) was obtained by freeze-drying the solvent.
1H NMR (300 MHz, D2O) δ3.96 (d, J=2.49 Hz, 2H), 3.80-3.71 (m, 1H), 3.64 (s, 2H), 3.35-3.20 (m, 2H), 3.01 (t, J=2.56 Hz, 1H), 2.68-2.54 (m, 5H), 2.17-2.05 (m, 2H); HRMS(ES+) calcd for C11H21N4O3 (M++H) 257.1614. found 257.1616.
(2) Synthesis of Example Compound 26
Figure US09371274-20160621-C00090
Synthesized by the same method as example compound 25 using compound 32b (43.2 mg, 71.8 μmol) (white solid, 18.7 mg, 38.3 μmol, two steps, 53%).
1H NMR (300 MHz, D2O) δ3.76-3.72 (m, 1H), 3.65 (s, 2H), 3.25-3.08 (m, 2H), 3.02 (t, J=7.44 Hz, 2H), 2.67-2.54 (m, 5H), 2.20-2.00 (m, 2H), 1.75-1.62 (m, 2H), 0.95 (t, J=7.56 Hz, 3H); HRMS(ES+) calcd for C11H25N4O3 (M++H) 261.1927. found 261.1931.
(3) Synthesis of Example Compound 27
Figure US09371274-20160621-C00091
Synthesized by the same method as example compound 25 using compound 33c (39.2 mg, 62.2 μmol) (white solid, 11.4 mg, 22.0 μmol, two steps, 35%).
1H NMR (300 MHz, D2O) δ3.76-3.67 (m, 1H), 3.56 (s, 2H), 3.19-3.08 (m, 2H), 3.02 (t, J=7.63 Hz, 2H), 2.66-2.51 (m, 5H), 2.14-2.02 (m, 2H), 1.67-1.60 (m, 2H), 1.35-1.22 (m, 4H), 0.85 (t, J=6.89 Hz, 3H); HRMS(ES+) calcd for C13H29N4O3 (M++H) 289.2240. found 289.2238.
(4) Synthesis of Example Compound 28
Figure US09371274-20160621-C00092
Synthesized by the same method as example compound 25 using compound 33d (35.2 mg, 54.2 μmol) (white solid, 9.23 mg, 17.2 μmol, two steps, 32%).
1H NMR (300 MHz, D2O) δ7.49-7.43 (m, 5H), 4.24 (s, 2H), 3.72-3.68 (m, 1H), 3.56 (s, 2H), 3.20-3.13 (m, 2H), 2.62-2.49 (m, 5H), 2.11-2.05 (m, 2H); HRMS(ES+) calcd for C15H25N4O3 (M++H) 309.1927. found 309.1920.
Example 10 Synthesis of Example Compounds 29-31
Example compounds 29-31 were synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00093
(A) Compounds 34a-34c (1) Synthesis of Compound 34a
Figure US09371274-20160621-C00094
10% Pd/C (5.49 mg) was stirred for 1 hour 30 minutes under a hydrogen atmosphere in a methanol solution (2 mL) of compound 30 (54.9 mg, 0.137 mmol). The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. N-Boc-glycine (26.5 mg, 0.151 mmol) and HOBt.H2O (23.1 mg, 0.151 mmol) were added to a DMF solution (2 mL) of the residue, triethylamine (22.8 μL, 0.164 mmol) and EDC.HCl (31.5 mg, 0.164 mmol) were added while cooling by ice and stirring, and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. After washing the organic layer sequentially with saturated sodium bicarbonate aqueous solution, water, and saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (57.6 mg, 0.108 mmol, two steps, 79%) was obtained by refining the residue obtained by silica gel column chromatography (chloroform: methanol=80:1).
HRMS(ES+) calcd for C24H46N5O8 (M++H) 532.3346. found 532.3329.
(2) Synthesis of Compound 34b
Figure US09371274-20160621-C00095
Synthesized by the same method as compound 34a using compound 30 (55.2 mg, 0.138 mmol) and N-Boc-y-aminobutyric acid (30.9 mg, 0.152 mmol) (white solid, 64.0 mg, 0.114 mmol, two steps, 83%).
HRMS(ES+) calcd for C26H50N5O8 (M++H) 560.3659. found 560.3646.
(3) Compound 34c
Figure US09371274-20160621-C00096
Synthesized by the same method as compound 34a using compound 30 (48.1 mg, 0.120 mmol) and N-Boc-ε-aminocaproic acid (30.5 mg, 0.132 mmol) (white solid, 52.5 mg, 89.4 μmol, two steps, 74%).
HRMS(ES+) calcd for C28H54N5O8 (M++H) 588.3972. found 588.3979.
(B) Example Compounds 29-31 (1) Synthesis of Example Compound 29
Figure US09371274-20160621-C00097
4 M hydrochloric acid/dioxane (2 mL) was added while cooling by ice and stirring to compound 34a (33.7 mg, 63.4 μmol) and stirred for one hour at room temperature. After distilling off the reaction solution under reduced pressure, the residue was dissolved in water and refined by HPLC. A colorless solid (13.9 mg, 27.5 μmol, 43%) was obtained by freeze drying the solvent.
1H NMR (300 MHz, D2O) δ3.80 (s, 2H), 3.66-3.57 (m, 3H), 3.41-3.28 (m, 2H), 2.68-2.50 (m, 5H), 1.94-1.80 (m, 2H); HRMS(ES+) calcd for C10H22N5O4 (M++H) 276.1672. found 276.1666.
(2) Synthesis of Example Compound 30
Figure US09371274-20160621-C00098
Synthesized by the same method as example compound 29 using compound 34b (32.4 mg, 57.9 μmol) (colorless solid, 17.3 mg, 32.5 μmol, 56%).
1H NMR (300 MHz, D2O) δ3.66-3.56 (m, 3H), 3.35-3.21 (m, 2H), 2.99 (t, J=7.73 Hz, 2H), 2.68-2.50 (m, 5H), 2.36 (t, J=7.51 Hz, 2H), 1.97-1.80 (m, 4H); HRMS(ES+) calcd for C12H26N5O4 (M++H) 304.1985. found 304.1982.
(3) Synthesis of Example Compound 31
Figure US09371274-20160621-C00099
Synthesized by the same method as example compound 29 using compound 34c (33.3 mg, 56.7 μmol) (colorless solid, 17.5 mg, 31.2 μmol, 55%).
1H NMR (300 MHz, D2O) δ3.66 (s, 2H), 3.65-3.53 (m, 1H), 3.38-3.21 (m, 2H), 2.97 (t, J=7.48 Hz, 2H), 2.68-2.50 (m, 5H), 2.26 (t, J=7.44 Hz, 2H), 1.91-1.78 (m, 2H), 1.70-1.55 (m, 4H), 1.40-1.30 (m, 2H); HRMS(ES+) calcd for C14H30N5O4 (M++H) 332.2298. found 332.2289.
Example 11 Synthesis of Example Compound 32
Example compound 32 was synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00100
(1) Synthesis of Compound 36
Figure US09371274-20160621-C00101
10% Pd/C (5.36 mg) was stirred for 2 hours 30 minutes under a hydrogen atmosphere in a methanol solution (2 mL) of compound 30 (53.6 mg, 0.134 mmol). The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. Triethylamine (22.3 μL, 0.161 mmol) and methanesulfonyl chloride (11.4 mg, 0.147 mmol) were added while cooling by ice and stirring to a dichloromethane solution (2 mL) of the residue and stirred for one hour at room temperature. Water was added to the reaction solution while cooling by ice and stirring, followed by extraction by chloroform. After washing the organic layer by saturated saline and drying (Na2SO4), the solvent was distilled off under reduced pressure. A colorless oily substance (43.1 mg, 95.3 μmol, two steps, 71%) was obtained by refining the residue obtained by silica gel column chromatography (chloroform:methanol=100:1).
HRMS(ES+) calcd for C18H37N4O7S (M++H) 453.2383. found 453.2390.
(2) Synthesis of Example Compound 32
Figure US09371274-20160621-C00102
4 M hydrochloric acid/dioxane (2 mL) was added while cooling by ice and stirring to compound 36 (22.1 mg, 48.9 μmol) and stirred for one hour at room temperature. After distilling off the reaction solution under reduced pressure, the residue was dissolved in water and refined by HPLC. A white solid (9.86 mg, 24.0 μmol, 49%) was obtained by freeze drying the solvent.
1H NMR (300 MHz, D2O) 63.75-3.69 (m, 1H), 3.63 (s, 2H), 3.24-3.13 (m, 2H), 3.05 (s, 3H), 2.66-2.48 (m, 5H), 1.97-1.83 (m, 2H); HRMS(ES+) calcd for C9H21N4O5S (M++H) 297.1233. found 297.1221.
Example 12 Synthesis of Example Compounds 33-46
Example compounds 33-46 were synthesized by the following synthesis scheme. In the scheme, the numbers appended below the structural formulas represent the compound numbers.
Figure US09371274-20160621-C00103
(A) Synthesis of Compound 38
Figure US09371274-20160621-C00104
Synthesized by the same method as compound 30 using compound 29 (100 mg, 0.367 mmol) and H2N—N(Me)CH2CO2Bn (143 mg, 0.734 mmol) (colorless oily substance, 127 mg, 0.291 mmol, two steps, 79%).
HRMS(ES+) calcd for C20H30N6O5Na (M++Na) 457.2175. found 457.2175.
(B) Compounds 39a-39n (1) Synthesis of Compound 39a
Figure US09371274-20160621-C00105
4 M hydrochloric acid/dioxane (2.0 mL) was added while cooling by ice to compound 38 (53.6 mg, 0.123 mmol) and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was used in the next reaction without refining. The residue was dissolved in DMF (2 mL), and Boc-Phe-OH (65.4 mg, 0.246 mmol) and HOBt.H2O (37.8 mg, 0.246 mmol) were added. Triethylamine (34.2 μL, 0.246 mmol) and EDC.HCl (47.3 mg, 0.246 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline, and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid (68.5 mg, 0.118 mmol, two steps, 96%) was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=80:1).
HRMS(ES+) calcd for C29H40N7O6 (M++H) 582.3040. found 582.3030.
(2) Synthesis of Compound 39b
Figure US09371274-20160621-C00106
Synthesized by the same method as compound 39a using compound 38 (42.8 mg, 98.6 μmol) (white solid, 49.5 mg, 82.9 μmol, two steps, 84%).
HRMS(ES+) calcd for C29H39N7O7Na (M++Na) 620.2809. found 620.2811.
(3) Synthesis of Compound 39c
Figure US09371274-20160621-C00107
Synthesized by the same method as compound 39a using compound 38 (44.3 mg, 0.102 mmol) (white solid, 53.2 mg, 99.8 μmol, two steps, 98%).
HRMS(ES+) calcd for C25H40N7O6 (M++H) 534.3040. found 534.3029.
(4) Synthesis of Compound 39d
Figure US09371274-20160621-C00108
Synthesized by the same method as compound 39a using compound 38 (109 mg, 0.422 mmol) (yellow oily substance, 38.0 mg, 75.2 μmol, two steps, 18%).
1H NMR (300 MHz, CDCl3) δ7.88 (s, 1H), 7.40-7.33 (m, 5H), 7.25-7.10 (m, 1H), 5.18 (s, 2H), 5.15-4.99 (m, 1H), 4.40-4.07 (m, 1H), 4.07-3.99 (m, 1H), 3.82-3.44 (m, 2H), 3.34 (t, J=6.23 Hz, 2H), 3.02-2.81 (m, 3H), 2.80-2.70 (m, 3H), 2.64-2.50 (m, 1H), 2.44-2.23 (m, 1H), 2.03-2.67 (m, 2H), 1.44 (s, 9H).
(5) Synthesis of Compound 39e
Figure US09371274-20160621-C00109
Synthesized by the same method as compound 39a using compound 38 (46.2 mg, 0.106 mmol) (yellow oily substance, 42.7 mg, 89.7 μmol, two steps, 84%).
HRMS(ES+) calcd for C22H34N7O6 (M++H) 492.2571. found 492.2576.
(6) Synthesis of Compound 39f
Figure US09371274-20160621-C00110
Synthesized by the same method as compound 39a using compound 38 (52.8 mg, 0.122 mmol) (white solid, 58.5 mg, 0.107 mmol, two steps, 88%).
HRMS(ES+) calcd for C26H42N7O6 (M++H) 548.3197. found 548.3201.
(7) Synthesis of Compound 39g
Figure US09371274-20160621-C00111
Synthesized by the same method as compound 39a using compound 38 (50.0 mg, 0.115 mmol) (white solid, 30.0 mg, 48.4 μmol, two steps, 42%).
1H NMR (300 MHz, CDCl3) δ8.21 (s, 1H), 7.42-7.27 (m, 5H), 5.88-5.76 (brs, 1H), 5.49-5.10 (m, 4H), 4.40-4.22 (m, 1H), 4.22-4.04 (m, 1H), 3.90-3.40 (m, 4H), 3.34 (t, J=6.16 Hz, 2H), 3.03-2.55 (m, 4H), 2.43-2.19 (m, 1H), 1.90-1.71 (m, 2H), 1.45 (s, 18H).
(8) Synthesis of Compound 39h
Figure US09371274-20160621-C00112
Synthesized by the same method as compound 39a using compound 38 (50.7 mg, 0.117 mmol) (white solid, 25.9 mg, 40.8 μmol, two steps, 35%).
1H NMR (300 MHz, CDCl3) δ7.86 (s, 1H), 7.42-7.27 (m, 5H), 5.42-5.01 (m, 5H), 4.45-4.23 (m, 1H), 4.16-4.00 (m, 1H), 3.83-3.56 (m, 2H), 3.46-3.24 (m, 2H), 3.14-2.97 (m, 2H), 2.97-2.51 (m, 4H), 2.44-2.24 (m, 1H), 2.10-1.57 (m, 4H), 1.44 (s, 18H).
(9) Synthesis of Compound 39i
Figure US09371274-20160621-C00113
Synthesized by the same method as compound 39a using compound 38 (51.4 mg, 0.118 mmol) (white solid, 74.3 mg, 0.115 mmol, two steps, 97%).
HRMS(ES+) calcd for C30H49N8O8 (M++H) 649.3673. found 649.3679.
(10) Synthesis of Compound 39j
Figure US09371274-20160621-C00114
Synthesized by the same method as compound 39a using compound 38 (50.9 mg, 0.117 mmol) (yellow solid, 72.3 mg, 0.109 mol, two steps, 93%).
HRMS(ES+) calcd for C31H51N8O8 (M++H) 663.3830. found 663.3823.
(11) Synthesis of Compound 39k
Figure US09371274-20160621-C00115
Synthesized by the same method as compound 39a using compound 38 (50.0 mg, 0.115 mmol) (white solid, 41.0 mg, 74.8 μmol, two steps, 65%).
HRMS(ES+) calcd for C24H37N8O7(M++H) 549.2785. found 549.2785.
(12) Synthesis of Compound 39l
Figure US09371274-20160621-C00116
Synthesized by the same method as compound 39a using compound 38 (50.2 mg, 0.116 mmol) (white solid, 47.9 mg, 74.9 μmol, two steps, 65%).
HRMS(ES+) calcd for C31H42N7O8 (M++H) 640.3095. found 640.3099.
(13) Synthesis of Compound 39m
Figure US09371274-20160621-C00117
Synthesized by the same method as compound 39a using compound 38 (50.9 mg, 0.117 mmol) (white solid, 54.9 mg, 97.6 μmol, two steps, 83%).
1H NMR (300 MHz, CDCl3) δ8.11 (s, 1H), 7.80-7.42 (m, 1H), 7.42-7.27 (m, 5H), 7.02-6.74 (m, 1H), 6.39-6.10 (m, 1H), 5.78-5.43 (m, 1H), 5.18 (s, 2H), 4.50-4.22 (m, 1H), 4.18-3.90 (m, 1H), 3.82-3.55 (m, 2H), 3.36 (s, 2H), 2.88-2.61 (m, 3H), 2.45-1.70 (m, 8H), 1.43 (s, 9H).
(14) Synthesis of Compound 39n
Figure US09371274-20160621-C00118
Synthesized by the same method as compound 39a using compound 38 (52.7 mg, 0.121 mmol) (white solid, 54.7 mg, 86.1 μmol, two steps, 71%).
HRMS(ES+) calcd for C32H44N7O8 (M++H) 654.3251. found 654.3262.
(C) Example Compounds 33-46 (1) Synthesis of Example Compound 33
Figure US09371274-20160621-C00119
10% Pd/C (4.2 mg) was added to a methanol (2 mL) solution of compound 39a (42.4 mg, 72.9 μmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for one hour at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 4 M hydrochloric acid/dioxane (2 mL) was added to the residue while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A white solid (10.4 mg, 17.5 μmol, two steps, 24%) was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 10-15% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV).
HRMS(ES+) calcd for C17H28N5O4 (M++H) 366.2141. found 366.2141.
(2) Synthesis of Example Compound 34
Figure US09371274-20160621-C00120
Synthesized by the same method as example compound 33 using compound 39b (30.0 mg, 50.2 μmol) (white solid, 5.12 mg, 8.95 μmol, two steps, 16%).
HRMS(ES+) calcd for C17H28N5O5 (M++H) 382.2090. found 382.2089.
(3) Synthesis of Example Compound 35
Figure US09371274-20160621-C00121
Synthesized by the same method as example compound 33 using compound 39c (33.4 mg, 62.6 μmol) (yellow solid, 4.07 mg, 7.47 μmol, two steps, 12%).
HRMS(ES+) calcd for C13H28N5O4 (M++H) 318.2141. found 318.2142.
(4) Synthesis of Example Compound 36
Figure US09371274-20160621-C00122
Synthesized by the same method as example compound 33 using compound 39d (29.8 mg, 58.9 μmol) (yellow solid, 11.2 mg, 21.6 μmol, two steps, 38%).
HRMS(ES+) calcd for C11H24N5O4 (M++H) 290.1828. found 290.1819.
(5) Synthesis of Example Compound 37
Figure US09371274-20160621-C00123
Synthesized by the same method as example compound 33 using compound 39e (24.7 mg, 51.9 μmol) (green solid, 7.50 mg, 14.9 μmol, two steps, 29%).
HRMS(ES+) calcd for C10H22N5O4 (M++H) 276.1672. found 276.1675.
(6) Synthesis of Example Compound 38
Figure US09371274-20160621-C00124
Synthesized by the same method as example compound 33 using compound 39f (32.0 mg, 58.5 μmol) (yellow solid, 13.4 mg, 23.9 μmol, two steps, 41%).
HRMS(ES+) calcd for C14H30N5O4 (M++H) 332.2298. found 332.2291.
(7) Synthesis of Example Compound 39
Figure US09371274-20160621-C00125
Synthesized by the same method as example compound 33 using compound 39g (30.0 mg, 48.4 μmol) (yellow solid, 9.20 mg, 14.2 μmol, two steps, 29%).
HRMS(ES+) calcd for C11H25N6O4 (M++H) 305.1937. found 305.1930.
(8) Synthesis of Example Compound 40
Figure US09371274-20160621-C00126
Synthesized by the same method as example compound 33 using compound 39h (25.9 mg, 40.8 μmol) (yellow solid, 4.96 mg, 7.51 μmol, two steps, 18%).
HRMS(ES+) calcd for C12H27N6O4 (M++H) 319.2094. found 319.2085.
(9) Synthesis of Example Compound 41
Figure US09371274-20160621-C00127
Synthesized by the same method as example compound 33 using compound 39i (44.6 mg, 68.8 μmol) (yellow solid, 3.35 mg, 4.97 μmol, two steps, 7.2%).
HRMS(ES+) calcd for C13H29N6O4 (M++H) 333.2250. found 333.2238.
(10) Synthesis of Example Compound 42
Figure US09371274-20160621-C00128
Synthesized by the same method as example compound 33 using compound 39j (57.8 mg, 87.3 μmol) (yellow solid, 11.89 mg, 17.3 μmol, two steps, 20%).
HRMS(ES+) calcd for C14H31N6O4 (M++H) 347.2407. found 347.2408.
(11) Synthesis of Example Compound 43
Figure US09371274-20160621-C00129
Synthesized by the same method as example compound 33 using compound 39k (34.6 mg, 63.1 μmol) (yellow solid, 10.64 mg, 19.0 μmol, two steps, 30%).
HRMS(ES+) calcd for C12H25N6O5 (M++H) 333.1886. found 333.1880.
(12) Synthesis of Example Compound 44
Figure US09371274-20160621-C00130
Synthesized by the same method as example compound 33 using compound 39l (34.4 mg, 53.8 μmol) (green solid, 9.56 mg, 17.0 μmol, two steps, 32%).
HRMS(ES+) calcd for C12H24N5O6 (M++H) 334.1727. found 334.1724.
(13) Synthesis of Example Compound 45
Figure US09371274-20160621-C00131
Synthesized by the same method as example compound 33 using compound 39m (48.2 mg, 85.7 μmol) (yellow solid, 20.5 mg, 35.7 μmol, two steps, 42%).
HRMS(ES+) calcd for C13H27N6O5 (M++H) 347.2043. found 347.2040.
(14) Synthesis of Example Compound 46
Figure US09371274-20160621-C00132
Synthesized by the same method as example compound 33 using compound 39n (44.8 mg, 70.5 μmol) (green solid, 18.55 mg, 32.3 μmol, two steps, 46%).
HRMS(ES+) calcd for C13H26N5O6 (M++H) 348.1883. found 348.1873.
Example 13 Synthesis of Example Compound 47 (1) Synthesis of Compound 40 ((7S,10S)-2-bromobenzyl 7-(3-((tert-butoxycarbonyl)amino)propyl)-10-isobutyl-3,14,14-trimethyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate)
Figure US09371274-20160621-C00133
10% Pd/C (10.6 mg) was added to a methanol (2 mL) solution of compound 14 (106 mg, 0.158 mmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for 35 minutes at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. Triethylamine (43.8 μL, 0.316 mmol) and (Boc)2O (69.0 mg, 0.316 mmol) were added to a DMF (2 mL) solution of the residue while cooling by ice and stirred for 2 hours 30 minutes at room temperature. 1 M hydrochloric acid was added to the reaction solution while cooling by ice, followed by extraction by ethyl acetate. The organic layer was washed with saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. o-Bromobenzyl alcohol (35.5 mg, 0.190 mmol) was added to a DMF (2 mL) solution of the residue obtained. N,N-dimethyl-4-aminopyridine (1.93 mg, 15.8 μmol) and N,N′-dicyclohexylcarbodiimide (35.9 mg, 0.174 mmol) were added while cooling by ice and stirred for 3 hours 20 minutes at room temperature. After distilling off the reaction solvent under reduced pressure, chloroform was added, and filtration was performed. The organic layer was washed with water and saturated saline and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=100:1). (60.08 mg, 85.2 μmol, three steps, 54%).
1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.59 (d, J=7.9 Hz, 1H), 7.45-7.29 (m, 3H), 7.23 (t, J=6.3 Hz, 1H), 7.05 (brs, 1H), 5.35-5.20 (m, 2H), 5.03-4.90 (m, 1H), 4.80-4.63 (m, 1H), 4.35-4.12 (m, 1H), 4.12-3.96 (m, 1H), 3.81-3.51 (m, 2H), 3.26-3.03 (m, 2H), 2.97-2.43 (m, 4H), 2.39-2.20 (m, 1H), 1.78-1.50 (m, 6H), 1.43 (s, 18H), 0.93 (s, 6H); HRMS(ES+) calcd for C32H52N5O8NaBr [M++Na] 736.2897. found 736.2919.
(2) Synthesis of Example Compound 47 (2-bromobenzyl 2-(2-((S)-6-amino-3-((S)-2-amino-4-methylpentanamido)hexanoyl)-1-methylhydrazinyl)acetate)
Figure US09371274-20160621-C00134
4 M hydrochloric acid/dioxane (2 mL) was added to compound 40 (40.6 mg, 56.9 μmol) while cooling by ice and stirred for one hour at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A white solid was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 25-45% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (21.7 mg, 29.3 μmol, 51%).
1H NMR (400 MHz, D2O) δ 8.35 (d, J=9.0 Hz, 1H), 7.69 (d, J=7.9 Hz, 1H), 7.55-7.47 (m, 1H), 7.43 (t, J=7.4 Hz, 1H), 7.33 (t, J=7.5 Hz, 1H), 5.30 (s, 2H), 4.30-4.24 (m, 1H), 4.02-3.85 (m, 1H), 3.80-3.60 (m, 2H), 3.07-2.90 (m, 2H), 2.64 (s, 3H), 2.34 (dd, J=15 and 5.2 Hz, 1H), 2.23 (dd, J=15 and 8.7 Hz, 1H), 1.80-1.44 (m, 7H), 0.93 (d, J=5.8 Hz, 6H); 13C NMR (100 MHz, D2O) δ 173.6, 173.0, 172.8, 137.2, 135.9, 133.9, 133.6, 131.0, 126.3, 69.9, 61.4, 54.8, 49.6, 47.3, 43.2, 41.9, 41.7, 33.6, 26.8, 26.4, 24.8, 23.7; HRMS(ES+) calcd for C22H37N5O4Br [M++H]+ 514.2029. found 514.2032.
Example 14 Synthesis of Example Compound 48 (1) Synthesis of Compound 41
Compound 41 ((S),(Z)-2-((tert-butoxycarbonyl)amino)undec-3-enoic acid) was synthesized with reference to the Journal of Peptide Synthesis, 19, 470-476, 2013.
(2) Synthesis of Compound 42 ((7S,10S)-benzyl 7-(2-azidoethyl)-3,14,14-trimethyl-10-((Z)-non-1-en-1-yl)-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate)
Figure US09371274-20160621-C00135
4 M hydrochloric acid/dioxane (2 mL) was added to compound 41 (90.0 mg, 0.207 mmol) while cooling by ice and stirred for 1 hour 15 minutes at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was used in the next reaction without refining. The residue was dissolved in DMF (2 mL), and (S),(Z)-2-((tert-butoxycarbonyl)amino)undec-3-enoic acid (0.372 mmol) and HOBt.H2O (57.0 mg, 0.372 mmol) were added. Triethylamine (51.8 μL, 0.372 mmol) and EDC.HCl (71.3 mg, 0.372 mmol) were added sequentially while cooling by ice and stirred overnight at room temperature. The reaction solution was added to 10% citric acid aqueous solution and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated saline, and dried by Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and a white solid was obtained by refining the residue obtained by silica gel chromatography (chloroform:methanol=100:1). (109 mg, 0.176 mmol, two steps, 85%).
1H NMR (400 MHz, CDCl3) δ7.76 (s, 1H), 7.43-7.33 (m, 5H), 5.62-5.52 (m, 1H), 5.45-5.22 (m, 1H), 5.18 (s, 2H), 4.95 (brs, 1H), 4.41-4.19 (m, 1H), 4.11-3.96 (m, 1H), 3.80-3.49 (m, 2H), 3.40-3.29 (m, 2H), 3.04-2.23 (m, 7H), 2.09-1.70 (m, 3H), 1.44 (s, 9H), 1.39-1.18 (m, 10H), 0.88 (t, J=6.6 Hz, 3H); HRMS(ES+) calcd for C31H50N7O6 [M++H]+ 616.3823. found 616.3813.
(3) Synthesis of Example Compound 48 (2-(2-((S)-5-amino-3-((S)-2-aminoundecanamido)pentanoyl)-1-methylhydrazinyl)acetic acid)
Figure US09371274-20160621-C00136
PtO2 (8.8 mg) was added to a methanol (2 mL) solution of compound 42 (18.8 mg, 30.5 μmol) under an argon atmosphere, hydrogen substitution was performed, and the solution was stirred for two hours at room temperature. The reaction solution was filtered by Celite, and the solvent was distilled off under reduced pressure. The residue obtained was used in the next reaction without refining. 4 M hydrochloric acid/dioxane (2 mL) was added to the residue while cooling by ice and stirred for 30 minutes at room temperature. The solvent was distilled off under reduced pressure, and the residue obtained was dissolved in water. A white cottony substance was obtained by refining by high-performance liquid chromatography (solvent, H2O in 0.1% TFA: CH3CN in 0.1% TFA. A linear gradient of 40-50% CH3CN in 0.1% TFA over 40 min. Flow rate 5 mL/min, detected at 222 nm UV). (5.52 mg, 8.77 μmol, two steps, 29%)
1H NMR (400 MHz, D2O) δ4.40-4.26 (m, 1H), 3.98-3.90 (m, 1H), 3.73-3.57 (m, 2H), 3.13-2.94 (m, 2H), 2.75-2.66 (m, 3H), 2.52 (dd, J=5.2 and 15 Hz, 1H), 2.40 (dd, J=8.7 and 15 Hz, 1H), 2.07-1.75 (m, 4H), 1.40-1.16 (m, 14H), 0.85 (t, J=7.0 Hz, 3H); HRMS (ES+) calcd for C19H40N5O4 [M++H]+ 402.3080. found 402.3098.
Example 15 Synthesis of Example Compound 49
Figure US09371274-20160621-C00137
Example compound 49 was synthesized by the same method as compound 24a, example compound 20 using compound 22a (100 mg, 0.197 mmol) and 3-chlorobenzyl alcohol (34.4 mg, 0.242 mmol) (white solid, 14.0 mg, three steps, 49%).
1H NMR (400 MHz, D2O) δ7.49-7.30 (m, 4H), 5.18 (s, 2H), 3.76-3.60 (m, 3H), 3.08 (t, J=8.6 Hz, 2H), 2.65 (s, 3H), 2.61-2.47 (m, 2H), 2.15-1.82 (m, 2H); HRMS(ES+) calcd for C15H24N4O3Cl [M++H]+343.1537. found 343.1535.
B. Measurement of Read-Through Activity Example 16 In Vitro Evaluation (1) Cell Culture
African green monkey SV40-transformed kidney fibroblast cell line (COS-7) was sown in 100×20 mm cell culture dishes manufactured by Falcon and cultured at 37° C. in 5% CO2. Fifty milliliters of fetal bovine serum (FBS) was added to 500 mL of D-MEM (containing high glucose, L-glutamine, and phenol red) manufactured by Wako Pure Chemical Industries Ltd., and culture broth prepared to adjust to 10% FBS was used as the culture broth. (Referred to hereinafter as culture broth.)
(2) Evaluation of Read-Through Activity
The Reporter Lysis Buffer 5×, assay 2× buffer, 1 M Na2CO3, and standard used in creation of the calibration curve referred to hereinafter are included in the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer manufactured by Promega.
COS-7 cells (8.0×103 cells/well) were seeded by continuous dispensing of 100 μL each into flat-bottom 96-well plates (Costar (registered trade mark) 3596) manufactured by Corning and incubated for 15-16 hours at 37° C.
A quantity of 4 μL/well of DNA solution prepared in a ratio of 1.32 μL (2 μg) of reporter gene (a construct obtained by linking a β-galactosidase gene and a luciferase gene and inserting a premature stop codon (TGA) at their juncture), 100 μL of OPTI-MEM (registered trade mark) I (Reduced Serum Medium 1×) manufactured by Invitrogen, and 4 μL of FuGENE (registered trade mark) HD Transfection Reagent manufactured by Roche was added to the solution and incubated for 10-11 hours at 37° C.
The entire culture broth was aspirated, and the compound to be evaluated was prepared to adjust to 200 μM by culture broth and added by continuously dispensing 200 μL/well. 100 μM and 50 μM were also prepared in the same way and added by continuously dispensing 200 μL each. Incubation was carried out for 48 hours at 37° C. after addition.
After 48 hours had elapsed, the entire culture broth in the well was aspirated and washed twice with PBS. The Reporter Lysis Buffer 5× was then diluted by MilliQ, and a solution prepared to adjust to Reporter Lysis Buffer l× was added by continuously dispensing 100 μL each and allowed to stand for 15 minutes at room temperature. The contents of each well were recovered thereafter, and the entire amount was transferred to Nunc microwell plates (V bottom) manufactured by Thermo Fisher Scientific and centrifuged (1800 rpm, 15 minutes). Eighty-five microliters of this lysate was transferred to Nunc low-binding 96-well plates (flat bottom) clear manufactured by Thermo Fisher Scientific.
<Evaluation of β-Galactosidase Activity>
Using Nunc low-binding 96-well plates (flat bottom) clear manufactured by Thermo Fisher Scientific, 30 μL/well of Reporter Lysis Buffer l× was added by dispensing continuously, followed by addition of 20 μL of the recovered lysate to make 50 μL. Separately, the standard required for creation of a calibration curve was added to make 0, 1, 2, 3, 4, and 5 mU according to the protocol of the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer manufactured by Promega. A quantity of 50 μL of Assay 2× buffer was added to each of these wells and incubated for 25 minutes at room temperature. A quantity of 150 μL/well of 1M Na2CO3, a reaction-stopping solution, was added, and the emission intensity (420 nm, reference 0 nm) was measured immediately using an absorption/fluorescence microplate reader Tecan Safire manufactured by Tecan. A calibration curve was created from the absorbance of the standard, and the absorbance converted to mU according to this calibration curve was taken as the activity value.
<Evaluation of Luciferase Activity>
A quantity of 50 μL/well of the recovered lysate was added to 96-well white plates (Costar (registered trade mark) 3912) manufactured by Corning and incubated for five minutes at room temperature after adding 100 μL of PicaGene (registered trade mark) manufactured by Toyo Ink Co., Ltd. After five minutes, the emission intensity was measured by a Berthold luminometer MicroLumat Plus LB96V manufactured by Berthold Japan.
<Calculation of Read-Through Efficiency>
The value obtained by dividing the luciferase activity by the β-galactosidase activity measured above was used.
The read-through activity was evaluated using example compound 2 synthesized in Example 1, example compound 3 synthesized in Example 5, and example compound 1 and example compound 4 synthesized separately by the same method. The results are shown in FIG. 1. It is understood from FIG. 1 that example compounds 1-4 have read-through activity in the same way as negamycin, and that example compound 3 in particular has remarkably high read-through efficiency.
Next, the read-through activity of example compound 5, obtained by esterifying the end carboxyl group of example compound 2, and example compound 7, obtained by introducing an N-linked amino acid group at position 3 of example compound 2, was evaluated. FIG. 2 shows the results obtained by evaluating the read-through activity of these compounds, example compound 2, example compound 3, and negamycin. FIG. 2 shows that example compounds 2, 3, 5, and 7 have remarkably high read-through efficiency in comparison even to negamycin.
The read-through activity of example compound 3 included in general formula (3) and example compound 12, obtained by introducing an N-linked amino acid group at position 3 thereof, was also evaluated. FIG. 3 shows that these compounds which have fewer carbon atoms in the main chain than example compound 2 also have read-through efficiency equal to or greater than that of negamycin.
Example 17
The read-through activity of example compounds 14-46 obtained in Examples 6-12 was evaluated by the same method as in Example 16. The results are shown below in Table 1.
TABLE 1
Example Compound Read-through Activity
14 2.13 ± 0.08
15 1.92 ± 0.21
16 1.87 ± 0.29
17 2.10 ± 0.90
18 1.30 ± 0.09
19 3.25 ± 0.07
20 7.07 ± 0.45
21 7.04 ± 0.14
22 5.18 ± 0.04
23 5.65 ± 0.09
24 3.89 ± 0.16
25 1.26 ± 0.07
26 1.44 ± 0.04
27 1.64 ± 0.12
28 1.40 ± 0.04
29 2.84 ± 0.06
30 1.70 ± 0.06
31 1.75 ± 0.02
32 1.24 ± 0.09
33 2.34 ± 0.11
34 2.00 ± 0.13
35 3.09 ± 0.62
36 1.92 ± 0.40
37 2.78 ± 0.67
38 1.75 ± 0.19
39 1.63 ± 0.14
40 1.50 ± 0.05
41 1.57 ± 0.05
42 2.14 ± 0.18
43 2.20 ± 0.05
44 1.58 ± 0.05
45 1.65 ± 0.18
46 1.60 ± 0.13
When negamycin was evaluated under the same conditions, the read-through activity was 1.53±0.04. As shown in Table 1, all of the example compounds of the present invention have read-through activity approximately equal to or greater than that of negamycin.
Example 18 In Vivo Evaluation
Transgenic mice were produced by introducing a luciferase gene and δ-galactosidase gene as reporter genes by the method described in International Publication WO2008/004610 pamphlet.
A physiological saline solution of example compound 2 synthesized in Example 1 was administered subcutaneously to the abdomen of the transgenic mice produced every 24 hours for seven consecutive days. On the eighth day, the rectus femoris and the gastrosoleus muscle of the mice were excised. After mincing by ophthalmological scissors, three times the wet weight of Reporter Lysis Buffer (Promega, US) was added, and the tissue was disrupted by Physcotron (Niti-on Co., Ltd.). After freezing overnight and thawing, the supernatant obtained by centrifugation was introduced into a 96-well plate, and the β-galactosidase activity was measured by a Beta-Glo Assay System (Promega, US) and the luciferase activity was measured by a Bright-Glo Assay System (Promega, US). A luminometer (ATTO) was used in measurement. The luciferase activity value was divided by the δ-galactosidase activity value, and the value multiplied by 104 was taken as the read-through efficiency.
FIG. 4 shows the results of measurement of the read-through activity of gentamicin, 5-epi-negamycin, and example compound 2 of the present invention. Example compound 2 of the present invention was found to have read-through activity and had high read-through activity even in comparison to gentamicin and 5-epi-negamycin.
Example 19
The read-through activity of example compounds 3, 7, 47, and 48 was evaluated by the same method as in Example 16. The results are shown in FIG. 5.
Here, Geneticin purchased from Roche was used as G418.
Example 20
In order to investigate the effects of the alkyl chain length of R12 of the N-linked amino acid residue represented by general formula (4), compounds (alkyl chains C4, C5, C7, and C11) of varied alkyl chain length of R12 in relationship to compound 48 were synthesized, and the read-through activity was evaluated by the same method as in Example 16. The results are shown in FIG. 6.
Furthermore, since products of the same molecular weight were obtained for C5 and C11 in FIG. 6, the measurement results of the two are shown (C5(1) and C5(2), C11(1) and C11 (2)).

Claims (13)

The invention claimed is:
1. A compound represented by formula (1):
Figure US09371274-20160621-C00138
in which
R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, and the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, or C6-C10 aralkenyl group,
one or both of R1 and R2 may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
X is N(R3)(R4), an N-linked amino acid residue, or a C1-C6 alkyl group containing an amino group having a substituent, where the substituent of the amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
R5 is a hydrogen, C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
R7 is a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of the substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, or a sulfonic acid group; and
n is an integer of 0-3,
or a pharmaceutically acceptable salt or solvate of the compound.
2. The compound according to claim 1 represented by formula (3):
Figure US09371274-20160621-C00139
h R1, R2, R3, R4, R5, R6, R7, and n are as defined in claim 1, or a salt or solvate of the compound.
3. The compound according to claim 1, wherein the N-linked amino acid residue of X is selected from α-amino acids and β-amino acids, or a pharmaceutically acceptable salt or solvate of the compound.
4. The compound according to claim 1 represented by formula (5):
Figure US09371274-20160621-C00140
in which R1, R2, X, R3, R4, R5, R6, and R7 are as defined in claim 1, or a pharmaceutically acceptable salt or solvate of the compound.
5. A compound represented by formula (1):
Figure US09371274-20160621-C00141
wherein at least one of R1 and R2 are an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded, the remaining R1 or R2 is hydrogen, and the amino acid residue is represented by the following formula (2):
Figure US09371274-20160621-C00142
X is N(R3)(R4), an N-linked amino acid residue, or a C1-C6 alkyl group containing an amino group having a substituent, where the substituent of the amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, and wherein the N-linked amino acid residue is an α-amino acid residue or β-amino acid residue comprising a C1-C12 side chain, and the α-amino acid is selected from the group consisting of isoleucine, valine, lysine, ornithine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, lysine, and α-hydroxyleucine;
R5 is a hydrogen, C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of the substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, or a sulfonic acid group;
n is an integer of 0-3;
R8 is hydrogen, an optionally substituted C1-C10 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, where substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
R9, R10 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group,
R9 and R10 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group; and
m is an integer of 0-4,
or a pharmaceutically acceptable salt or solvate of the compound.
6. A compound represented by formula (1):
Figure US09371274-20160621-C00143
wherein
R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, and the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, or C6-C10 aralkenyl group,
one or both of R1 and R2 may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
R5 is a hydrogen, C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group;
R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of the substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, or a sulfonic acid group;
n is an integer of 0-3;
the N-linked amino acid residue of X is represented by the following formula (4):
Figure US09371274-20160621-C00144
in which
R11 is hydrogen or an optionally substituted C1-C6 alkyl group;
R12 is hydrogen, an optionally substituted C7-C9 alkyl group, C2-C10 alkenyl group, C2-C10 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group, where substituents that these substituents can have are a halogen, hydroxyl group, carboxyl group, amino group, or amide group;
R13, R14 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R8 and R9 also may form, together with the nitrogen atom to which they are bonded, a three- to six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members (for example, oxazolidine), where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, C6-C10 aralkyl group, C6-C10 aralkenyl group; and
p is an integer of 0-3,
or a pharmaceutically acceptable salt or solvate of the compound.
7. A method of inducing read-through activity through a nonsense mutation in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising a compound represented by the following general formula (1):
Figure US09371274-20160621-C00145
in which
R1 and R2 are each independently hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, sulfonyl group, cyclic amine, or guanidyl group,
R1 and R2 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group,
one or both of R1 and R2 may be an amino acid residue introduced via an amide bond formed together with the nitrogen atom to which they are bonded;
X is N(R3)(R4), an N-linked amino acid residue, or C1-C6 alkyl group containing an amino group having a substituent, where the substituent of this amino group is hydrogen, an optionally substituted C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, where R3 and R4 are each independently hydrogen or an optionally substituted C1-C6 alkyl group, R3 and R4 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
R5 is a hydrogen, C1-C4 alkyl group, C2-C4 alkenyl group, or C2-C4 alkynyl group;
R6 is a C1-C4 alkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, C3-C6 cycloalkyl group, carboxyl group, carboxylate group, alkyl group having a carboxyl group or carboxylate group; C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C10 aralkyl group, C6-C10 aralkenyl group, cyclic amine, or guanidyl group, R5 and R6 also may form, together with the nitrogen atom to which they are bonded, a five-membered or six-membered heterocycle or heteroaryl bonded via N, may also contain 1-3 additional hetero atoms selected from the group consisting of O, N, and S as ring constituent members, where the heterocycle or heteroaryl may be substituted by a C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group, C6-C10 aralkyl group, or C6-C10 aralkenyl group;
R7 is hydrogen, a C1-C6 alkyl group, substituted or unsubstituted aralkyl group, or C3-C7 cycloalkyl group, where the substituent of a substituted aralkyl group is a halogen, C1-C4 alkyl group, alkoxy group, hydroxy group, nitro group, amino group, C1-C6 acyl group, or amino group modified by an alkyl group, or sulfonic acid group; and
n is an integer of 0-3,
or a pharmaceutically acceptable salt or solvate of the compound.
8. The method of claim 7, wherein the compound is represented by the following general formula (5):
Figure US09371274-20160621-C00146
in which R1, R2, X, R3, R4, R5, R6, and R7 are as defined in claim 7, or a pharmaceutically acceptable salt or solvate of the compound.
9. The method according to claim 7 or 8 wherein the subject has a disease caused by a nonsense mutation.
10. The method of claim 7, wherein the subject has a disease selected from the group consisting of muscular dystrophy, Duchenne muscular dystrophy, infantile neuronal ceroid lipofuscinosis, multiple sclerosis, Alzheimer's disease, Tay-Sachs disease, neurodegenerative disease, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis, hemophilia, von Willebrand disease, ataxia telangiectasia, β-thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler disease, familial hypercholesterolemia, cerebellar ataxia, nodular sclerosis, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial cholesterolemia, pigmentary retinopathy, amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, Niemann-Pick disease, and Marfan syndrome.
11. The method of claim 10, wherein the disease is selected from the group consisting of muscular dystrophy, cystic fibrosis, Hurler disease, and infantile neuronal ceroid lipofuscinosis.
12. A compound selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt or solvate of the compound:
Figure US09371274-20160621-C00147
Figure US09371274-20160621-C00148
Figure US09371274-20160621-C00149
Figure US09371274-20160621-C00150
Figure US09371274-20160621-C00151
Figure US09371274-20160621-C00152
Figure US09371274-20160621-C00153
Figure US09371274-20160621-C00154
Figure US09371274-20160621-C00155
Figure US09371274-20160621-C00156
Figure US09371274-20160621-C00157
Figure US09371274-20160621-C00158
Figure US09371274-20160621-C00159
Figure US09371274-20160621-C00160
Figure US09371274-20160621-C00161
Figure US09371274-20160621-C00162
Figure US09371274-20160621-C00163
Figure US09371274-20160621-C00164
Figure US09371274-20160621-C00165
Figure US09371274-20160621-C00166
Figure US09371274-20160621-C00167
Figure US09371274-20160621-C00168
Figure US09371274-20160621-C00169
Figure US09371274-20160621-C00170
Figure US09371274-20160621-C00171
Figure US09371274-20160621-C00172
Figure US09371274-20160621-C00173
Figure US09371274-20160621-C00174
Figure US09371274-20160621-C00175
Figure US09371274-20160621-C00176
Figure US09371274-20160621-C00177
Figure US09371274-20160621-C00178
Figure US09371274-20160621-C00179
Figure US09371274-20160621-C00180
Figure US09371274-20160621-C00181
Figure US09371274-20160621-C00182
Figure US09371274-20160621-C00183
Figure US09371274-20160621-C00184
Figure US09371274-20160621-C00185
Figure US09371274-20160621-C00186
Figure US09371274-20160621-C00187
Figure US09371274-20160621-C00188
Figure US09371274-20160621-C00189
Figure US09371274-20160621-C00190
13. A compound selected from the group consisting of the following compounds 52 and 53, or a pharmaceutically acceptable salt or solvate of the compound:
Figure US09371274-20160621-C00191
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